Low sweel tissue adhesive and sealant formulations

ABSTRACT

A hydrogel tissue adhesive formed by reacting an aldehyde-functionalized dextran containing pendant aldehyde groups with a multi-arm polyethylene glycol amine is described. The hydrogel exhibits little to no swell upon exposure to physiological conditions. The hydrogel may be useful as a tissue adhesive or sealant for medical applications that require a low swell hydrogel to inhibit complications, such as fibrosis, including scar formation or surgical adhesions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/859,458, filed Jul. 29, 2013 the contents of which are incorporatedby reference herein.

FIELD OF THE INVENTION

The present disclosure relates to the field of medical adhesives. Morespecifically, the present disclosure relates to a hydrogel tissueadhesive formed by reacting an aldehyde-functionalized dextrancontaining pendant aldehyde groups with a multi-arm polyethylene glycolamine.

BACKGROUND OF THE INVENTION

Tissue adhesives have many potential medical applications, includingwound closure, supplementing or replacing sutures or staples in internalsurgical procedures, preventing leakage of fluids such as blood, bile,gastrointestinal fluid and cerebrospinal fluid, adhesion of syntheticonlays or inlays to the cornea, drug delivery devices, and asanti-adhesion barriers to prevent post-surgical adhesions. Conventionaltissue adhesives are generally not suitable for a wide range of adhesiveapplications. For example, cyanoacrylate-based adhesives have been usedfor topical wound closure, but the release of toxic degradation productslimits their use for internal applications. Fibrin-based adhesives areslow curing, have poor mechanical strength, and pose a risk of viralinfection. Additionally, fibrin-based adhesives do not bond covalentlyto the underlying tissue.

Several types of hydrogel tissue adhesives have been developed, whichhave improved adhesive and cohesive properties and are nontoxic (see,for example, Sehl et al., U.S. Patent Application Publication No.2003/0119985, and Goldmann, U.S. Patent Application Publication No.2005/0002893). These hydrogels are generally formed by reacting acomponent having nucleophilic groups with a component havingelectrophilic groups, which are capable of reacting with thenucleophilic groups of the first component, to form a crosslinkednetwork via covalent bonding. However, these hydrogels typically swell,dissolve away too quickly, or lack sufficient adhesion or mechanicalstrength, thereby decreasing their effectiveness as surgical adhesives.

SUMMARY OF THE INVENTION

The present disclosure is directed to a hydrogel tissue adhesive andsealant that has good adhesion and cohesion properties, crosslinksreadily at body temperature, does not degrade rapidly, is nontoxic tocells and non-inflammatory to tissue, and maintains dimensionalstability better and longer than traditional oxidizedpolysaccharide-based hydrogel tissue adhesives.

In one embodiment, the present disclosure provides a kit for forming alow swell hydrogel comprising a first aqueous solution or dispersioncomprising one or more aldehyde-functionalized dextrans containingpendant aldehyde groups, said aldehyde-functionalized dextrans having aweight-average molecular weight of about 10,000 to about 20,000 Daltonsand an equivalent weight per aldehyde group of about 226 (degree ofaldehyde substitution of about 90%) to about 170 (degree of aldehydesubstitution of about 120%); and a second aqueous solution or dispersioncomprising one or more polyethylene glycols having eight arms,substantially each arm of which is terminated with at least one primaryamine group, wherein the polyethylene glycols have a number-averagemolecular weight of about 9,000 to about 11,000 Daltons; wherein (i) thetotal concentration of the aldehyde-functionalized dextrans containingpendant aldehyde groups in the first aqueous solution or dispersion isabout 5 wt % to about 20 wt % and the total concentration of thepolyethylene glycols in the second aqueous solution or dispersion isabout 10 wt % to about 18 wt %; or (ii) the total concentration of thealdehyde-functionalized dextrans containing pendant aldehyde groups inthe first aqueous solution or dispersion is about 5 wt % to about 10 wt% and the total concentration of the polyethylene glycols in the secondaqueous solution or dispersion is about 10 wt % to about 20 wt %.

In another embodiment, the present disclosure provides a dried hydrogelformed by a process comprising the steps of combining in a solvent oneor more aldehyde-functionalized dextrans containing pendant aldehydegroups, said aldehyde-functionalized dextrans having a weight-averagemolecular weight of about 10,000 to about 20,000 Daltons and anequivalent weight per aldehyde group of about 226 (degree of aldehydesubstitution of about 90%) to about 170 (degree of aldehyde substitutionof about 120%), and one or more polyethylene glycols having eight arms,substantially each arm of the which is terminated with at least oneprimary amine group, said polyethylene glycols having a number-averagemolecular weight of about 9,000 to about 11,000 Daltons, to form a lowswell hydrogel; wherein (i) the total concentration of thealdehyde-functionalized dextrans containing pendant aldehyde groups inthe solvent is about 5 wt % to about 20 wt % and the total concentrationof the polyethylene glycols in the solvent is about 10 wt % to about 18wt %; or (ii) the total concentration of the aldehyde-functionalizeddextrans containing pendant aldehyde groups in the solvent is about 5 wt% to about 10 wt % and the total concentration of the polyethyleneglycols in the solvent is about 10 wt % to about 20 wt %; and treatingsaid hydrogel to remove at least a portion of said solvent to form thedried hydrogel.

In another embodiment, the present disclosure provides a compositioncomprising the reaction product of at least one aldehyde-functionalizeddextran containing pendant aldehyde groups, wherein thealdehyde-functionalized dextran has a weight-average molecular weight ofabout 10,000 to about 20,000 Daltons and an equivalent weight peraldehyde group of about 226 (degree of aldehyde substitution of about90%) to about 170 (degree of aldehyde substitution of about 120%), andat least one polyethylene glycol having eight arms, substantially eacharm of which is terminated with at least one primary amine group,wherein the polyethylene glycol has a number-average molecular weight ofabout 9,000 to about 11,000 Daltons; wherein (i) the compositioncontains about 5 wt % to about 20 wt % of the aldehyde-functionalizeddextran and about 10 wt % to about 18 wt % of the polyethylene glycol;or the composition contains about 5 wt % to about 10 wt % of thealdehyde-functionalized dextran and about 10 wt % to about 20 wt % ofthe polyethylene glycol.

In another embodiment, the present disclosure provides a crosslinkedhydrogel composition comprising at least one aldehyde-functionalizeddextran containing pendant aldehyde groups, wherein thealdehyde-functionalized dextran has a weight-average molecular weight ofabout 10,000 to about 20,000 Daltons and an equivalent weight peraldehyde group of about 226 (degree of aldehyde substitution of about90%) to about 170 (degree of aldehyde substitution of about 120%), andat least one polyethylene glycol having eight arms, substantially eacharm of which is terminated with at least one primary amine group,wherein the polyethylene glycol has a number-average molecular weight ofabout 9,000 to about 11,000 Daltons; wherein (i) the compositioncontains about 5 wt % to about 20 wt % of the aldehyde-functionalizeddextran and about 10 wt % to about 18 wt % of the polyethylene glycol;or (ii) the composition contains about 5 wt % to about 10 wt % of thealdehyde-functionalized dextran and about 10 wt % to about 20 wt % ofthe polyethylene glycol; and wherein said aldehyde-functionalizeddextran and said polyethylene glycol are crosslinked through covalentbonds formed between the pendant aldehyde groups of dextran and theprimary amine groups of the polyethylene glycol.

Finally, in another embodiment, the present disclosure provides a methodfor applying a coating to an anatomical site on tissue of a livingorganism comprising the steps of applying to the site (a)aldehyde-functionalized dextrans containing pendant aldehyde groups,wherein the aldehyde-functionalized dextrans have a weight-averagemolecular weight of about 10,000 to about 20,000 Daltons and anequivalent weight per aldehyde group of about 226 (degree of aldehydesubstitution of about 90%) to about 170 (degree of aldehyde substitutionof about 120%); followed by (b) polyethylene glycols having eight arms,substantially each arm of which is terminated with at least one primaryamine group, wherein the polyethylene glycols have a number-averagemolecular weight of about 9,000 to about 11,000 Daltons, or (b) followedby (a), or premixing (a) and (b) and applying the resulting mixture tothe site before the resulting mixture completely cures; and wherein theweight percent ratio of aldehyde-functionalized dextrans to polyethyleneglycols is about 2:1 to about 1:4.

DETAILED DESCRIPTION

The present disclosure is related to compositions (e.g., hydrogels)formed by reacting an aldehyde-functionalized dextran containing pendantaldehyde groups with a water-dispersible, multi-arm polyethylene glycolamine. The compositions may be useful as tissue adhesives or sealantsfor medical applications that require a tissue adhesive or sealant thatexhibits little or no swell when exposed to physiological conditions.

As used above and throughout the description of the disclosure, thefollowing terms, unless otherwise indicated, shall be defined asfollows:

The term “aldehyde-functionalized dextran(s)” as used herein, refers toa dextran that has been chemically modified to introduce pendantaldehyde groups into the molecule. In many instances, the terms dextranand dextrans are used interchangeably. The pendant aldehyde groups maybe single aldehyde groups or dialdehydes. As defined herein,aldehyde-functionalized dextran does not include dextran that isoxidized by cleavage of the dextran ring to introduce aldehyde groups.Oxidation of the dextran ring results in dialdehydes formed by openingthe rings of dextran.

The term “pendant aldehyde group” refers to an aldehyde group that isattached to dextran via one of the ring hydroxyl groups.

The term “degree of aldehyde substitution” refers to the mole percent ofpendant aldehyde groups per mole of repeat units, i.e., (moles ofpendant aldehyde groups/moles of dextran repeat units)×100. The degreeof aldehyde substitution is calculated as described in the Examplesherein.

The term “equivalent weight per aldehyde group” refers to the molecularweight of dextran divided by the number of pendant aldehyde groupsintroduced into the molecule.

The term “multi-arm polyethylene glycol amine” refers to a polyethyleneglycol (PEG) polymer having three or more polyethylene glycol chains(“arms”), which may be linear or branched, emanating from a centralstructure, which may be a single atom, a core molecule, or a polymerbackbone, wherein at least three of the branches (“arms”) are terminatedby at least one primary amine group. The multi-arm polyethylene glycolamine is water soluble or is able to be dispersed in water to form acolloidal suspension capable of reacting with a second reactant inaqueous solution or dispersion.

The term “dispersion” as used herein, refers to a colloidal suspensioncapable of reacting with a second reactant in an aqueous medium.

The term “branched” refers to a polyethylene glycol polymer having oneor more branch points (“arms”), including star, dendritic, comb, highlybranched, and hyperbranched polyethylene glycol polymers. Branchesradiate from one or more trifunctional or higher functional branchpoints.

The term “dendritic” refers to a highly branched polymer having abranching structure that repeats regularly with each successivegeneration of monomer radiating from a core molecule.

The term “comb polymer” refers to a branched polymer in which linearside-chains emanate from trifunctional branch points on a linear polymerbackbone.

The term “star polymer” refers to a branched polymer in which linearside-chains emanate from a single atom or a core molecule having a pointof symmetry.

The term “hyperbranched polymer” refers to a highly branched polymerwhich is more branched than “highly branched,” with order approachingthat of an imperfect dendrimer.

The term “highly branched polymer” refers to a branched polymer havingmany branch points, such that the distance between branch points issmall relative to the total length of arms.

The term “primary amine” refers to a neutral amino group having two freehydrogens. The amino group may be bound to a primary, secondary ortertiary carbon.

The term “multi-functional amine” refers to a chemical compoundcomprising at least two functional groups, at least one of which is aprimary amine group.

The term “crosslink” refers to a bond or chain of atoms attached betweenand linking two different polymer chains.

The term “crosslink density” is herein defined as the reciprocal of theaverage number of chain atoms between crosslink connection sites.

The term “% by weight”, also referred to herein as “wt %”, refers to theweight percent relative to the total weight of the solution ordispersion, unless otherwise specified.

The term “anatomical site” refers to any external or internal part ofthe body of humans or animals.

The term “tissue” refers to any biological tissue, both living and dead,in humans or animals.

The term “hydrogel” refers to a water-swellable polymeric matrix,consisting of a three-dimensional network of macromolecules heldtogether by covalent crosslinks that can absorb a substantial amount ofwater to form an elastic gel.

The term “dried hydrogel” refers to a hydrogel that has been treated toremove at least a portion of the solvent contained therein. Preferably,substantially all of the solvent is removed from the hydrogel.

The term “low swell” refers to hydrogels which exhibit a % swell of lessthan about 10%, more particularly about 5%, more particularly about 2%,more particularly about 1%, and even more particularly about 0% asmeasured by the hydrolysis method described in the Examples. The methodis an in-vivo model wherein swell is the percentage increase in thelength of the hydrogel relative to its initial length. In someembodiments, the hydrogels exhibit a negative swell, i.e. the hydrogelsshrink. The present disclosure also embodies these hydrogels exhibitinga negative swell.

The term “M_(w)” as used herein refers to the weight-average molecularweight.

The term “M_(n)” as used herein refers to the number-average molecularweight.

The term “M_(z)” as used herein refers to the z-average molecularweight.

The term “medical application” refers to medical applications as relatedto humans and animals.

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliter(s), “L” means liter(s), “μL” means microliter(s), “cm” meanscentimeter(s), “mm” means millimeter(s), “μm” means micrometer(s), “mol”means mole(s), “mmol” means millimole(s), “g” means gram(s), “mg” meansmilligram(s), “mol %” means mole percent, “Vol” means volume, “w/w”means weight per weight, “Da” means Daltons, “kDa” means kiloDaltons,the designation “10K” means that a polymer molecule possesses anumber-average molecular weight of 10 kiloDaltons, “M” means molarity,“kPa” means kilopascals, “psi” means pounds per square inch, “rpm” meansrevolutions per minute”, “¹H NMR” means proton nuclear magneticresonance spectroscopy, “13-C NMR” means carbon 13 nuclear magneticresonance spectroscopy, “ppm” means parts per million, “cP” meanscentipoise, “PBS” means phosphate-buffered saline, “MWCO” meansmolecular weight cut off.

A reference to “Aldrich” or a reference to “Sigma” means the saidchemical or ingredient was obtained from Sigma-Aldrich, St. Louis, Mo.

Aldehyde-Functionalized Dextran

Aldehyde-functionalized dextran suitable for use herein is dextran thathas been chemically modified to introduce pendant aldehyde groups intothe molecule. The pendant aldehyde groups may be single aldehyde groupsor dialdehydes. The pendant aldehyde groups of thealdehyde-functionalized dextran disclosed herein are attached to dextranthrough linking groups. In one embodiment, the linking groups comprisecarbon, hydrogen, and oxygen atoms, but do not contain a nitrogen atom.In one embodiment, the linking groups are attached to dextran by etherlinkages. Aldehyde-functionalized dextran having these types of linkinggroups are more stable in aqueous solution than oxidized polysaccharidesor aldehyde-functionalized polysaccharides having other types of linkinggroups, such as those that contain a nitrogen atom or are linked to thepolysaccharide by other chemical linkages (e.g., amide or urethane).These more stable dextrans are, therefore, more practical for commercialpurposes. In one embodiment, the linking groups are attached to dextranby ester linkages. The ester linkages may be used to provide a fasterdegrading hydrogel. In one embodiment, the linking group contains analkoxy group alpha to the pendant aldehyde group (i.e., on an adjacentcarbon atom). In another embodiment, the linking group does not containan alkoxy group beta to the pendant aldehyde group (i.e., on the secondcarbon atom from the aldehyde group).

As used herein, aldehyde-functionalized dextran does not include dextranthat is oxidized by cleavage of the dextran ring to introduce aldehydegroups. Oxidation of the dextran ring results in dialdehydes formed byopening the rings of dextran. Therefore, the dialdehyde groups formed byoxidation of dextran rings are not pendant aldehyde groups as definedherein.

Aldehyde-functionalized dextran may be prepared by chemically modifyingdextran to introduce pendant aldehyde groups. Dextran is availablecommercially from sources such as Sigma Chemical Co. (St. Louis, Mo.).Typically, dextran is a heterogeneous mixture having a distribution ofdifferent molecular weights, and are characterized by an averagemolecular weight, for example, the weight-average molecular weight(M_(w)), or the number average molecular weight (M_(n)), as is known inthe art. Therefore, the aldehyde-functionalized dextran prepared fromthese dextrans are also a heterogeneous mixture having a distribution ofdifferent molecular weights. Suitable aldehyde-functionalized dextranhas a weight-average molecular weight of about 10,000 to about 20,000Daltons, more particularly about 13,000 to about 17,000 Daltons, moreparticularly about 14,000 to about 16,000 Daltons, and more particularlyabout 15,000 Daltons. In one embodiment, the aldehyde-functionalizeddextran has a weight-average molecular weight of about 15,000 Daltons.

Aldehyde-functionalized dextran may be prepared using methods known inthe art. Aldehyde-functionalized dextran may be prepared using any ofthe methods described by Mehta et al. (WO 99/07744). For example,dextran may be reacted with allyl glycidyl ether in an acid aqueousmedium to form allyloxy dextran which is then oxidized by ozonolysis tocleave the double bond and introduce a terminal aldehyde group, asdescribed in detail in the Examples herein below. Additionally, glycidolmay be reacted with a dextran in a basic aqueous medium to give analkylated dextran, as described by Chen (Biotechnology Techniques3:131-134, 1989). Periodate oxidation of the alkylated dextran yields analdehyde-functionalized dextran having pendant aldehyde groups. Thealdehyde-functionalized dextran may also be prepared by the methoddescribed by Solarek et al. (U.S. Pat. No. 4,703,116) wherein dextran isreacted with a derivatizing acetal reagent in the presence of base andthen the acetal is hydrolyzed by adjusting the pH to less than 7.0.

Aldehyde-functionalized dextran having dialdehyde functional groups canbe prepared by first attaching a pendant group containing either aterminal diene or by attaching a cyclic, disubstituted olefin to thedextran ring. Attachment of the pendant groups can be accomplished usinga variety of methods, including reaction of dextran with glycidyl etherscontaining cyclic olefins or terminal dienes, or reaction withcarboxylic acids or derivatives thereof which also contain cyclicolefins or terminal dienes. Oxidation of dextran derivatized with cyclicolefins or terminal dienes using methods known in the art, such asozonolysis, yield dextran derivatized with pendant dialdehydes.

The equivalent weight per aldehyde group and the degree of aldehydesubstitution may be determined using methods known in the art, asdescribed in detail in the Examples herein. Suitablealdehyde-functionalized dextran has an equivalent weight per aldehydegroup of about 226 (degree of aldehyde substitution of about 90%) toabout 170 (degree of aldehyde substitution of about 120%), moreparticularly about 226 (degree of aldehyde substitution of about 90%) toabout 185 (degree of aldehyde substitution of about 110%), moreparticularly about 222 (degree of aldehyde substitution of about 92%) toabout 189 (degree of aldehyde substitution of about 108%), moreparticularly about 219 (degree of aldehyde substitution of about 93%) toabout 190 (degree of aldehyde substitution of about 107%), moreparticularly about 217 (degree of aldehyde substitution of about 94%) toabout 192 (degree of aldehyde substitution of about 106%), moreparticularly about 212 (degree of aldehyde substitution of about 96%),to about 196 (degree of aldehyde substitution of about 104%), moreparticularly about 210 (degree of aldehyde substitution of about 97%),to about 190 (degree of aldehyde substitution of about 107%), moreparticularly about 204 (degree of aldehyde substitution of about 100%),to about 196 (degree of aldehyde substitution of about 104%), moreparticularly about 177 (degree of aldehyde substitution of about 115%).In one particular embodiment, the equivalent weight per aldehyde groupis about 216 (degree of aldehyde substitution about 94%). It is to beunderstood that any aldehyde-functionalized dextran equivalent weightper aldehyde group within the range of about 226 (degree of aldehydesubstitution of about 90%) to about 170 (degree of aldehyde substitutionof about 120%) can be useful in the present disclosure.

Multi-Arm Polyethylene Glycol Amines

Suitable multi-arm polyethylene glycol amines include compounds having3, 4, 6, or 8 arms terminated with primary amines (referred to herein as3, 4, 6, or 8-arm star PEG amines, respectively). In one embodiment, themulti-arm polyethylene glycol amine is an 8-arm PEG amine.

Multi-arm polyethylene glycol amines may have a number-average molecularweight of about 9,000 to about 11,000 Daltons, more particularly fromabout 9,500 to about 10,500 Daltons, and more particularly about 10,000Daltons. In one embodiment, the multi-arm polyethylene glycol amine isan 8-arm polyethylene glycol having eight arms terminated by a primaryamine group and having a number-average molecular weight of about 10,000Daltons.

The multi-arm polyethylene glycol amines are either availablecommercially or may be prepared using methods known in the art. Forexample, multi-arm polyethylene glycols, wherein substantially each armis terminated by a primary amine group, may be prepared by putting amineends on multi-arm polyethylene glycols (e.g., 3, 4, 6, and 8-arm starpolyethylene glycols, available from companies such as NektarTransforming Therapeutics; SunBio, Inc., Anyang City, South Korea; NOFCorp., Tokyo, Japan; or JenKem Technology USA, Allen, Tex.) using themethod described by Buckmann et al. (Makromol. Chem. 182:1379-1384,1981). In that method, the multi-arm polyethylene glycol is reacted withthionyl bromide to convert the hydroxyl groups to bromines, which arethen converted to amines by reaction with ammonia at 100° C.Additionally, multi-arm polyethylene glycol amines may be prepared frommulti-arm polyols using the method described by Chenault (commonly ownedU.S. Pat. No. 7,868,132). In that method, the multi-arm polyether isreacted with thionyl chloride to convert the hydroxyl groups to chlorinegroups, which are then converted to amines by reaction with aqueous oranhydrous ammonia. Other methods that may be used for preparingmulti-arm polyethylene glycol amines are described by Merrill et al. inU.S. Pat. No. 5,830,986, and by Chang et al. in WO 97/30103.

In one embodiment, the multi-arm polyethylene glycol amine is aneight-arm branched end polyethylene glycol amine having two primaryamine groups at the end of the polymer arms and having a number-averagemolecular weight of about 10,000 Daltons, as described by Arthur et al.(U.S. Pat. No. 8,282,959).

In another embodiment, the multi-arm polyethylene glycol amine is amixture of an eight-arm branched end polyethylene glycol amine havingtwo primary amine groups at the end of the polymer arms and having anumber-average molecular weight of about 10,000 Daltons, and aneight-arm polyethylene glycol amine having eight arms terminated by aprimary amine group and having a number-average molecular weight ofabout 10,000 Daltons.

It should be recognized that the water-dispersible, multi-armpolyethylene glycol amines are generally a somewhat heterogeneousmixture having a distribution of arm lengths and in some cases, adistribution of species with different numbers of arms. When a multi-armamine has a distribution of species having different numbers of arms, itcan be referred to based on the average number of arms in thedistribution. For example, in one embodiment the multi-arm amine is an8-arm star PEG amine, which comprises a mixture of multi-arm star PEGamines, some having less than and some having more than 8 arms; however,the multi-arm star PEG amines in the mixture have an average of 8 arms.Therefore, the terms “8-arm”, “6-arm”, “4-arm” and “3-arm” as usedherein to refer to multi-arm amines, should be construed as referring toa heterogeneous mixture having a distribution of arm lengths and in somecases, a distribution of species with different numbers of arms, inwhich case the number of arms recited refers to the average number ofarms in the mixture.

Methods of Using the Hydrogel Tissue Adhesive

The hydrogel tissue adhesive disclosed herein may be used in variousforms. In one embodiment, the aldehyde-functionalized dextran containingpendant aldehyde groups and the multi-arm polyethylene glycol amine areused as components of aqueous solutions or dispersions. To prepare anaqueous solution or dispersion comprising an aldehyde-functionalizeddextran (referred to herein as the “first aqueous solution ordispersion”), at least one aldehyde-functionalized dextran is added towater to give a concentration of about 5% to about 20%, moreparticularly from about 5% to about 15%, and more particularly fromabout 5% to about 10% by weight relative to the total weight of thesolution or dispersion. Additionally, a mixture of at least twodifferent aldehyde-functionalized dextrans having differentweight-average molecular weights, different degrees of aldehydesubstitution, or both different weight-average molecular weights anddegrees of aldehyde substitution may be used. Where a mixture ofaldehyde-functionalized polysaccharides is used, the total concentrationof the aldehyde-functionalized polysaccharides is about 5% to about 20%by weight, more particularly from about 5% to about 15%, and moreparticularly from about 5% to about 10% by weight relative to the totalweight of the solution or dispersion.

Similarly, to prepare an aqueous solution or dispersion comprising amulti-arm polyethylene glycol amine (referred to herein as the “secondaqueous solution or dispersion”), at least one water-dispersible,multi-arm polyethylene glycol amine (e.g., 8-arm PEG amine) is added towater to give a concentration of about 10% to about 20% by weight, moreparticularly from about 10% to about 18% by weight relative to the totalweight of the solution or dispersion. The optimal concentration to beused depends on the intended application and on the concentration of thealdehyde-functionalized dextran used in the first aqueous solution ordispersion. Additionally, a mixture of different multi-arm polyethyleneglycol amine having different number-average molecular weights,different numbers of arms, or both different number-average molecularweights and different numbers of arms may be used. Where a mixture ofmulti-arm polyethylene glycol amine is used, the total concentration ofthe multi-arm amines is about 10% to about 20% by weight, moreparticularly from about 10% to about 18% by weight relative to the totalweight of the solution or dispersion.

For use on living tissue, it is preferred that the first aqueoussolution or dispersion and the second aqueous solution or dispersion besterilized to prevent infection. Any suitable sterilization method knownin the art that does not adversely affect the ability of the componentsto react to form an effective hydrogel may be used, including, but notlimited to, electron beam irradiation, gamma irradiation, ethylene oxidesterilization, or filtration through a 0.2 μm pore membrane.

The first aqueous solution or dispersion and the second aqueous solutionor dispersion may further comprise various additives depending on theintended application. Preferably, the additive does not interfere witheffective gelation to form a hydrogel. The amount of the additive useddepends on the particular application and may be readily determined byone skilled in the art using routine experimentation. For example, thefirst aqueous solution or dispersion and/or the second aqueous solutionor dispersion may comprise at least one additive selected from pHmodifiers, antimicrobials, colorants, surfactants, pharmaceutical drugsand therapeutic agents.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one pH modifierto adjust the pH of the solution(s) or dispersion(s). Suitable pHmodifiers are well known in the art. The pH modifier may be an acidic orbasic compound. Examples of acidic pH modifiers include, but are notlimited to, carboxylic acids, inorganic acids, and sulfonic acids.Examples of basic pH modifiers include, but are not limited to,hydroxides, alkoxides, nitrogen-containing compounds other than primaryand secondary amines, and basic carbonates and phosphates.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one antimicrobialagent. Suitable antimicrobial preservatives are well known in the art.Examples of suitable antimicrobials include, but are not limited to,alkyl parabens, such as methylparaben, ethylparaben, propylparaben, andbutylparaben; triclosan; chlorhexidine; cresol; chlorocresol;hydroquinone; sodium benzoate; and potassium benzoate.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one colorant toenhance the visibility of the solution(s) or dispersion(s). Suitablecolorants include dyes, pigments, and natural coloring agents. Examplesof suitable colorants include, but are not limited to, FD&C and D&Ccolorants, such as FD&C Violet No. 2, FD&C Blue No. 1, D&C Green No. 6,D&C Green No. 5, D&C Violet No. 2; and natural colorants such asbeetroot red, canthaxanthin, chlorophyll, eosin, saffron, and carmine.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one surfactant.Surfactant, as used herein, refers to a compound that lowers the surfacetension of water. The surfactant may be an ionic surfactant, such assodium lauryl sulfate, or a neutral surfactant, such as polyoxyethyleneethers, polyoxyethylene esters, and polyoxyethylene sorbitan.

Additionally, the first aqueous solution or dispersion and/or the secondaqueous solution or dispersion may optionally include at least onepharmaceutical drug or therapeutic agent. Suitable drugs and therapeuticagents are well known in the art (for example see the United StatesPharmacopeia (USP), Physician's Desk Reference (Thomson Publishing), TheMerck Manual of Diagnosis and Therapy 18th ed., Mark H. Beers and RobertBerkow (eds.), Merck Publishing Group, 2006; or, in the case of animals,The Merck Veterinary Manual, 9th ed., Kahn, C. A. (ed.), MerckPublishing Group, 2005). Nonlimiting examples include anti-inflammatoryagents, for example, glucocorticoids such as prednisone, dexamethasone,budesonide; non-steroidal anti-inflammatory agents such as indomethacin,salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen;fibrinolytic agents such as a tissue plasminogen activator andstreptokinase; anti-coagulants such as heparin, hirudin, ancrod,dicumarol, sincumar, iloprost, L-arginine, dipyramidole and otherplatelet function inhibitors; antibodies; nucleic acids; peptides;hormones; growth factors; cytokines; chemokines; clotting factors;endogenous clotting inhibitors; antibacterial agents; antiviral agents;antifungal agents; anti-cancer agents; cell adhesion inhibitors; healingpromoters; vaccines; thrombogenic agents, such as thrombin, fibrinogen,homocysteine, and estramustine; radio-opaque compounds, such as bariumsulfate and gold particles and radiolabels.

Additionally, the second aqueous solution or dispersion comprising themulti-arm polyethylene glycol amine may optionally comprise at least oneother multi-functional amine having one or more primary amine groups toprovide other beneficial properties, such as hydrophobicity or modifiedcrosslink density. The multi-functional amine is capable of inducinggelation when mixed with an oxidized dextran in an aqueous solution ordispersion. The multi-functional amine may be a second multi-armpolyethylene glycol amine, such as those described above, or anothertype of multi-functional amine, including, but not limited to, linearand branched diamines, such as diaminoalkanes, polyaminoalkanes, andspermine; branched polyamines, such as polyethylenimine; cyclicdiamines, such as N,N′-bis(3-aminopropyl)piperazine,5-amino-1,3,3-trimethylcyclohexanemethylamine,1,3-bis(aminomethyl)cyclohexane, 1,4-diaminocyclohexane, andp-xylylenediamine; aminoalkyltrialkoxysilanes, such as3-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane;aminoalkyldialkoxyalkylsilanes, such as3-aminopropyldiethoxymethylsilane, dihydrazides, such as adipicdihydrazide; linear polymeric diamines, such as linear polyethylenimine,α,ω-amino-terminated polyethers, am-bis(3-aminopropyl)polybutanediol,β,ω-1-amino-terminated polyethers (linear Jeffamines); comb polyamines,such as chitosan, polyallylamine, and polylysine, and di- andpolyhydrazides, such as bis(carboxyhydrazido)polyethers andpoly(carboxyhydrazido) star polyethers. Many of these compounds arecommercially available from companies such as Sigma-Aldrich and HuntsmanLLC. Typically, if present, the multi-functional amine is used at aconcentration of about 5% by weight to about 1000% by weight relative tothe weight of the multi-arm polyethylene glycol amine in the aqueoussolution or dispersion.

When the first aqueous solution or dispersion and the second aqueoussolution or dispersion are mixed they react to form a crosslinkedhydrogel composition comprising at least one aldehyde-functionalizeddextran containing pendant aldehyde groups; and at least one multi-armpolyethylene glycol amine wherein substantially each arm of which isterminated with at least one primary amine group, and wherein thealdehyde-functionalized dextran and the multi-arm polyethylene glycolamine are crosslinked through covalent bonds formed between the pendantaldehyde groups of the aldehyde-functionalized dextran and the primaryamine groups of the multi-arm polyethylene glycol amine. The covalentbonds may be imine, aminal or hemiaminal bonds.

In one embodiment, the present disclosure relates to a compositioncomprising the reaction product of at least one aldehyde-functionalizeddextran containing pendant aldehyde groups, wherein thealdehyde-functionalized dextran has a weight-average molecular weight ofabout 10,000 to about 20,000 Daltons and an equivalent weight peraldehyde group of about 226 (a degree of aldehyde substitution of about90%) to about 170 (a degree of aldehyde substitution of about 120%), andat least one polyethylene glycol having eight arms, substantially eacharm of which is terminated with at least one primary amine group,wherein the polyethylene glycol has a number-average molecular weight ofabout 9,000 to about 11,000 Daltons; wherein (i) the compositioncontains about 5 wt % to about 20 wt % of the aldehyde-functionalizeddextran and about 10 wt % to about 18 wt % of the polyethylene glycol;or (ii) the composition contains about 5 wt % to about 10 wt % of thealdehyde-functionalized dextran and about 10 wt % to about 20 wt % ofthe polyethylene glycol.

In another embodiment, the present disclosure relates to a crosslinkedhydrogel composition comprising at least one aldehyde-functionalizeddextran containing pendant aldehyde groups, wherein thealdehyde-functionalized dextran has a weight-average molecular weight ofabout 10,000 to about 20,000 Daltons and an equivalent weight peraldehyde group of about 226 (a degree of aldehyde substitution of about90%) to about 170 (a degree of aldehyde substitution of about 120%), andat least one polyethylene glycol having eight arms, substantially eacharm of which is terminated with at least one primary amine group,wherein the polyethylene glycol has a number-average molecular weight ofabout 9,000 to about 11,000 Daltons, wherein (i) the compositioncontains about 5 wt % to about 20 wt % of the aldehyde-functionalizeddextran and about 10 wt % to about 18 wt % of the polyethylene glycol;or (ii) the composition contains about 5 wt % to about 10 wt % of thealdehyde-functionalized dextran and about 10 wt % to about 20 wt % ofthe polyethylene glycol; and wherein said aldehyde-functionalizeddextran and said polyethylene glycol are crosslinked through covalentbonds formed between the pendant aldehyde groups of dextran and theprimary amine groups of the polyethylene glycol.

The swelling of the hydrogel may be substantially reduced or eliminatedby using the prescribed amounts of the aldehyde-functionalized dextranin the first aqueous solution or dispersion and the multi-armpolyethylene amine in the second aqueous solution or dispersion in termsof weight percent and/or by altering the amount of functionalization ofeither component, as shown in the Examples herein below.

The first aqueous solution or dispersion and the second aqueous solutionor dispersion may be used to apply a coating to an anatomical site ontissue of a living organism. The two aqueous solutions or dispersionsmay be applied to the site in any number of ways. Once both solutions ordispersions are combined on a site, they crosslink to form a hydrogelwhich provides either a coating on the site, a barrier film, afiller/spacer or a sealant.

In one embodiment, the two aqueous solutions or dispersions are appliedto the site sequentially using any suitable means including, but notlimited to, spraying, brushing with a cotton swab or brush, or extrusionusing a pipette, or a syringe. The solutions or dispersions may beapplied in any order. Then, the solutions or dispersions are mixed onthe site using any suitable device, such as a cotton swab, a spatula, orthe tip of the pipette or syringe.

In another embodiment, the two aqueous solutions or dispersions aremixed manually before application to the site. The resulting mixture isthen applied to the site before it completely cures using a suitableapplicator, as described above.

In one embodiment, the present disclosure relates to a method forapplying a low swell coating to an anatomical site on tissue of a livingorganism comprising the steps of applying to the site (a)aldehyde-functionalized dextrans containing pendant aldehyde groups,wherein the aldehyde-functionalized dextrans have a weight-averagemolecular weight of about 10,000 to about 20,000 Daltons and anequivalent weight per aldehyde group of about 226 (a degree of aldehydesubstitution of about 90%) to about 170 (a degree of aldehydesubstitution of about 120%); followed by polyethylene glycols havingeight arms, substantially each arm of which is terminated with at leastone primary amine group, wherein the polyethylene glycols have anumber-average molecular weight of about 9,000 to about 11,000 Daltons.Alternatively, the method for applying the coating may be reversed suchthat the polyethylene glycols are applied first followed by thealdehyde-functionalized dextrans. The aldehyde-functionalized dextransand the polyethylene glycols may also be premixing to form a mixture andthen the resulting mixture is applied to the site before it completelycures.

The relative amounts of the aldehyde-functionalized dextrans and thepolyethylene glycols applied may be between about 2:1 (i.e. 20 wt %dextran to 5 wt % PEG) to about 1:4 (i.e. 5 wt % dextran to 20 wt %PEG). More particularly, the relative amounts of thealdehyde-functionalized dextrans and the polyethylene glycols appliedmay be between about 2:1 (i.e. 20 wt % dextran to 10 wt % PEG) to about5:18 (i.e. 5 wt % dextran to 18 wt % PEG). Even more particularly, therelative amounts of the aldehyde-functionalized dextrans and thepolyethylene glycols applied may be between about 1:1 (i.e. 10 wt %dextran to 10 wt % PEG) to about 1:4 (i.e. 5 wt % dextran to 20 wt %PEG). Even more particularly, the relative amounts of thealdehyde-functionalized dextrans and the polyethylene glycols appliedmay be between about 1:1 (i.e. 10 wt % dextran to 10 wt % PEG) to about5:18 (i.e. 5 wt % dextran to 18 wt % PEG).

For example, the method for applying a low swell coating to ananatomical site on tissue of a living organism may include the steps ofapplying to the site (a) aldehyde-functionalized dextrans containingpendant aldehyde groups, wherein the aldehyde-functionalized dextranshave a weight-average molecular weight of about 15,000 Daltons and anequivalent weight per aldehyde group of about 177 (a degree of aldehydesubstitution of about 115%); followed by polyethylene glycols havingeight arms, substantially each arm of which is terminated with at leastone primary amine group, wherein the polyethylene glycols have anumber-average molecular weight of about 10,000 Daltons, wherein theweight percent ratio of aldehyde-functionalized dextrans to polyethyleneglycols is about 1:1.5.

In another example, the method for applying a low swell coating to ananatomical site on tissue of a living organism may include the steps ofapplying to the site (a) aldehyde-functionalized dextrans containingpendant aldehyde groups, wherein the aldehyde-functionalized dextranshave a weight-average molecular weight of about 15,000 Daltons and anequivalent weight per aldehyde group of about 177 (a degree of aldehydesubstitution of about 115%); followed by polyethylene glycols havingeight arms, substantially each arm of which is terminated with at leastone primary amine group, wherein the polyethylene glycols have anumber-average molecular weight of about 10,000 Daltons, wherein theweight percent ratio of aldehyde-functionalized dextrans to polyethyleneglycols is about 1:1.

The first aqueous solution or dispersion and the second aqueous solutionor dispersion may be applied to the site simultaneously where they mixto form a hydrogel. For example, the two aqueous solutions ordispersions may be contained in separate barrels of a double-barrelsyringe. In this way the two aqueous solutions or dispersions areapplied simultaneously to the site with the syringe. Suitabledouble-barrel syringe applicators are known in the art. For example,Red1 describes several suitable applicators for use in the invention inU.S. Pat. No. 6,620,125, (particularly FIGS. 1, 5, and 6, which aredescribed in Columns 4, line 10 through column 6, line 47). The twoaqueous solutions or dispersions may also be applied to the site using adual-lumen catheter, such as those available from Bistech, Inc. (Woburn,Mass.). Additionally, injection devices for introducing two liquidcomponents endoscopically into the body simultaneously are known in theart and may be adapted for the delivery of the two aqueous solutions ordispersions disclosed herein (see for example, Linder et al., U.S. Pat.No. 5,322,510).

In another embodiment, the first aqueous solution or dispersion and thesecond aqueous solution or dispersion may be premixed and delivered tothe site using a double barrel syringe containing a motionless mixer,such as that available from ConProtec, Inc. (Salem, N.H.) or MixpacSystems AG (Rotkreuz, Switzerland). Other suitable mixers are describedby Ashmead et al. (U.S. Pat. Nos. 8,246,241 and 8,277,113; and U.S.Patent Application Publication Nos. 2012/0325854 and 2013/0020352).Alternatively, the mixing tip may be equipped with a spray head, such asthat described by Cruise et al. in U.S. Pat. No. 6,458,147.Additionally, the mixture of the two aqueous solutions or dispersionsfrom the double-barrel syringe may be applied to the site using acatheter or endoscope. Devices for mixing a two liquid component tissueadhesive and delivering the resulting mixture endoscopically are knownin the art and may be adapted for the mixing and delivery of the twoaqueous solutions or dispersions disclosed herein (see for example,Nielson, U.S. Pat. No. 6,723,067; and Red1 et al., U.S. Pat. No.4,631,055).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a spray device, such as those described byFukunaga et al. (U.S. Pat. No. 5,582,596), Delmotte et al. (U.S. Pat.No. 5,989,215) or Sawhney (U.S. Pat. No. 6,179,862) or Brunk et al.(U.S. Patent Application Publication Nos. 2012/0000935 and2012/0000993).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a minimally invasive surgical applicator, suchas those described by Sawhney (U.S. Pat. No. 7,347,850).

In another embodiment, the hydrogel tissue adhesive disclosed herein maybe used in the form of a dried hydrogel. In this embodiment, a driedhydrogel is prepared by combining in a solvent at least onealdehyde-functionalized dextran with at least one multi-arm polyethyleneglycol amine to form a hydrogel, and treating the hydrogel to remove atleast a portion of the solvent to form the dried hydrogel. Suitablesolvents include, but are not limited to, water, ethanol, isopropanol,tetrahydrofuran, hexanes, polyethylene glycol, and mixtures thereof. Iftwo different solvents are used, the two solvents are miscible with eachother. In one embodiment the solvent is water. Thealdehyde-functionalized dextran and multi-arm polyethylene glycol aminemay be combined in various ways. For example, the first aqueous solutionor dispersion comprising the aldehyde-functionalized dextran and thesecond aqueous solution or dispersion comprising the multi-armpolyethylene glycol amine, may be prepared and mixed as described aboveto form the hydrogel. The solutions or dispersions used to prepare thehydrogel may further comprise various additives depending on theintended application. Any of the additives described above may be used.The hydrogel is then treated to remove at least a portion of the solventcontained therein to form the dried hydrogel. Preferably, substantiallyall of the solvent is removed from the hydrogel. The solvent may beremoved from the hydrogel using methods known in the art, for example,using heat, vacuum, a combination of heat and vacuum, or flowing astream of dry air or a dry inert gas such as nitrogen over the hydrogel.The dried hydrogel may be sterilized using the methods described above.The dried hydrogel may be applied to an anatomical site in a number ofways, as described below. The dried hydrogel may be hydrated on the siteby the addition of a suitable aqueous solution such as water or a buffer(e.g., phosphate-buffered saline) or by the physiological fluids presentat the site.

In one embodiment, the present disclosure relates to a dried hydrogelformed by a process comprising the steps of combining in a solvent oneor more aldehyde-functionalized dextrans containing pendant aldehydegroups, said aldehyde-functionalized dextrans having a weight-averagemolecular weight of about 10,000 to about 20,000 Daltons and anequivalent weight per aldehyde group of about 226 (a degree of aldehydesubstitution of about 90%) to about 170 (a degree of aldehydesubstitution of about 120%), and one or more polyethylene glycols havingeight arms, substantially each arm of the which is terminated with atleast one primary amine group, said polyethylene glycols having anumber-average molecular weight of about 9,000 to about 11,000 Daltons,to form a hydrogel, wherein (i) the total concentration of thealdehyde-functionalized dextrans containing pendant aldehyde groups inthe solvent is about 5 wt % to about 20 wt % and the total concentrationof the polyethylene glycols in the solvent is about 10 wt % to about 18wt %; or (ii) the total concentration of the aldehyde-functionalizeddextrans containing pendant aldehyde groups in the solvent is about 5 wt% to about 10 wt % and the total concentration of the polyethyleneglycols in the solvent is about 10 wt % to about 20 wt %; and treatingsaid hydrogel to remove at least a portion of said solvent to form thedried hydrogel.

In one embodiment, the dried hydrogel may be used in the form of a film.The dried hydrogel film may be formed by casting a mixture of thesolutions or dispersions, as described above, on a suitable substrateand treating the resulting hydrogel to form a dried hydrogel film. Thedried hydrogel film may be applied directly to an anatomical site.Additionally, the dried hydrogel film may be used to bond two anatomicalsites together.

In another embodiment, the dried hydrogel may be used in the form offinely divided particles. The dried hydrogel particles may be formed bycomminuting the dried hydrogel using methods known in the art,including, but not limited to, grinding, milling, or crushing with amortar and pestle. The dried hydrogel particles may be applied to ananatomical site in a variety of ways, such as sprinkling or spraying,and may also be used to bond two anatomical sites together.

Kits

In one embodiment, the present invention relates to a kit for forming alow swell hydrogel including a first aqueous solution comprising one ormore aldehyde-functionalized dextrans containing pendant aldehydegroups, said aldehyde-functionalized dextrans having a weight-averagemolecular weight of about 10,000 to about 20,000 Daltons and anequivalent weight per aldehyde group of about 226 (a degree of aldehydesubstitution of about 90%) to about 170 (a degree of aldehydesubstitution of about 120%), more particularly about 226 (a degree ofaldehyde substitution of about 90%) to about 185 (a degree of aldehydesubstitution of about 110%); and a second aqueous solution comprisingone or more polyethylene glycols having eight arms, substantially eacharm of which is terminated with at least one primary amine group,wherein the polyethylene glycols have a number-average molecular weightof about 9,000 to about 11,000 Daltons; wherein (i) the totalconcentration of the aldehyde-functionalized dextrans containing pendantaldehyde groups in the first aqueous solution is about 5 wt % to about20 wt % and the total concentration of the polyethylene glycols in thesecond aqueous solution is about 10 wt % to about 18 wt %; or (ii) thetotal concentration of the aldehyde-functionalized dextrans containingpendant aldehyde groups in the first aqueous solution is about 5 wt % toabout 10 wt % and the total concentration of the polyethylene glycols inthe second aqueous solution is about 10 wt % to about 20 wt %. Each ofthe aqueous solutions or dispersions may be contained in any suitablevessel, such as a vial or a syringe barrel.

The total concentration of the aldehyde-functionalized dextranscontaining pendant aldehyde groups in the first aqueous solution mayalso be about 5 wt % to about 10 wt % and the total concentration of thepolyethylene glycols in the second aqueous solution may also be about 10wt % to about 18 wt %.

For example, the kit for forming a low swell hydrogel may include afirst aqueous solution comprising one or more aldehyde-functionalizeddextrans containing pendant aldehyde groups, saidaldehyde-functionalized dextrans having a weight-average molecularweight of about 15,000 Daltons and an equivalent weight per aldehydegroup of about 177 (a degree of aldehyde substitution of about 115%);and a second aqueous solution comprising one or more polyethyleneglycols having eight arms, substantially each arm of which is terminatedwith at least one primary amine group, wherein the polyethylene glycolshave a number-average molecular weight of about 10,000 Daltons; whereinthe total concentration of the aldehyde-functionalized dextranscontaining pendant aldehyde groups in the first aqueous solution isabout 10 wt % and the total concentration of the polyethylene glycols inthe second aqueous solution is about 15 wt %.

In another example, the kit for forming a low swell hydrogel may includea first aqueous solution comprising one or more aldehyde-functionalizeddextrans containing pendant aldehyde groups, saidaldehyde-functionalized dextrans having a weight-average molecularweight of about 15,000 Daltons and an equivalent weight per aldehydegroup of about 177 (a degree of aldehyde substitution of about 115%);and a second aqueous solution comprising one or more polyethyleneglycols having eight arms, substantially each arm of which is terminatedwith at least one primary amine group, wherein the polyethylene glycolshave a number-average molecular weight of about 10,000 Daltons; whereinthe total concentration of the aldehyde-functionalized dextranscontaining pendant aldehyde groups in the first aqueous solution isabout 15 wt % and the total concentration of the polyethylene glycols inthe second aqueous solution is about 15 wt %.

The kit may also include at least one aldehyde-functionalized dextrancontaining pendant aldehyde groups and at least one multi-armpolyethylene glycol amine in the form of finely divided powders, asdescribed above. The powders may be contained in separate containers orthey may be premixed and contained in a single container. The kit mayalso comprise an aqueous solution for hydrating the powders.

In another embodiment, the kit comprises a dried hydrogel as describedabove. The dried hydrogel may be in the form of a film, finely dividedparticles, or other dried forms. The kit may further comprise an aqueoussolution for hydrating the dried hydrogel. The dried hydrogel particlesmay be contained in any suitable container.

Medical Applications

The hydrogel disclosed herein may be useful as a tissue adhesive orsealant for medical applications that require a tissue adhesive orsealant that exhibits little or no swell (e.g., low swell) when exposedto physiological conditions. In these applications, thealdehyde-functionalized dextran and the multi-arm polyethylene glycolamine combination (e.g., hydrogel, composition, or dried hydrogel) maybe applied to the desired anatomical site using the methods describedabove.

In one embodiment, the present disclosure is directed to compositionshaving low swelling biocompatible polymers and methods using suchcompositions to inhibit fibrosis, including scar formation and surgicaladhesions. The methods of the present disclosure include applying thealdehyde-functionalized dextran and the multi-arm polyethylene glycolamine combination to tissue involved in or affected by a surgicalprocedure wherein fibrosis, including scar formation, keloid formationand surgical adhesions, may occur.

Surgical adhesions, which include the attachment of organs or tissues toeach other through scar tissue, can produce clinical problems. Theformation of scar tissue is well known in surgical procedures or othertissue injuries and is required for proper wound healing. There are somecases wherein the scar tissue overgrows the intended region and createssurgical adhesions. These scar tissue surgical adhesions restrict thenormal mobility and function of affected body parts. Where peripheralnerves are involved, fibrous adhesions may even elicit severe painduring normal movement.

For example, the formation of fibrous adhesions to the spinal cord durawhich occurs as part of the natural healing process after laminectomysurgery is commonly occurring problem which causes poor surgicaloutcomes, persistent pain to patient and often requires a secondsurgery. The aldehyde-functionalized dextran and the multi-armpolyethylene glycol amine hydrogel of the present disclosure is aneffective treatment or preventative measure in this surgical indication.The hydrogel provides low to zero swell at the application site toreduce or eliminate any increased pressure on the spinal cord. Thehydrogel persists at the application site for a sufficient time toprotect the dura from adhesion formation during normal soft tissuehealing (e.g., 7-10 days). The hydrogel is biodegradable and dissolveswithin weeks or months after application, depending on the individualcomposition of the hydrogel. The hydrogel contains monomer and/orcrosslinking units which are hydrolysable under physiologicalconditions, and are broken in the human or animal body.

A clinically important example of detrimental scar formation occurs withperidural fibrosis. This condition leads to recurrent low back pain andleg pain which can persist after lumbar laminectomy, laminotomy anddiscectomy. In this situation, scar formation restricts nerve rootmobility and has been associated with recurrent pain in the same areathat was treated.

A number of studies have been done to test various treatments forpreventing peridural fibrosis. For example, fat grafts have been usedwith some success to prevent or ameliorate scar formation (e.g., spinaladhesion prevention). Gelfoam (denatured collagen gel) and silasticmembranes have also showed some effectiveness in preventing adhesions.Later studies, however, indicated that Gelfoam was ineffective orpromoted scar formation. Sodium hyaluronate is known to retard fibrosisand reduced fibroblast invasion in dog models. Another related productis DuraSeal Xact (Covidien) which is a synthetic resorbable hydrogelthat was developed for use as a dural sealant to provide watertightclosure and to also allow for inhibition of peridural fibrosis. Anotherproduct is Oxiplex/SP Gel (FzioMed) which is a combination ofcarboxymethyl cellulose and polyethylene oxide used to coat exposedsurgical sites to prevent scarring.

The compositions and methods of the present disclosure are suitable fortreating animals, preferably mammals, and more preferably humans. Forexample, a therapeutically effective amount of the low swell hydrogel,or related composition, as disclosed herein can be safely administeredto treat a lesion in an animal to inhibit scar formation, and fibrosisin general.

In one embodiment, the compositions and methods of the presentdisclosure address a need in spinal surgery by providing a low swell,degradable material which allows for healing and also prevention of theformation of fibrous adhesions during the healing process. Adhesioncompromise the success of spinal decompression surgeries by causingadditional and sustainable pain and not allowing a reduction in pain tooccur after the procedure.

The compositions and methods of the present disclosure are useful, forone or more of the reasons addressed herein, in the followingprocedures.

Spinal surgeries, including lumbar laminectomy, lumbar discectomy,flexor tendon surgery, spinal fusion and joint replacement or repair. Inone embodiment, the present disclosure provides materials and methodsfor inhibiting fibrosis following laminectomy, in particular, inhibitingepidural (peridural) fibrosis following a lumbar laminectomy. Forexample, when applied to the site of the laminectomy the site may showreduced or minimal scar tissue formation and bone growth, and the duramater may be visible as a smooth transparent membrane.

Abdominal procedures, including surgeries of the intestines, appendix,cholecystectomy, hernial repair, lysis of peritoneal adhesions, kidney,bladder, urethra, and prostate.

Gynecological procedures, including surgeries to treat infertility dueto bilateral tubal disease with adhesion attached to ovaries, fallopiantubes and fimbriae, salingostomy, salpingolysis and ovariolysis. Theseprocedures include other gynecological surgeries, such as the removal ofendometriosis, preventing de-novo adhesion formation, treatment ofectopic pregnancy, myomectomy of uterus or fundus, and hysterectomy.

Musculoskeletal surgeries, including fibrosis of joints resulting fromtraumatic injury, such as a fall or collision, which may render theinjured joint stiff and movement painful, in part because scar tissuemay form in the traumatized area after tendon damage. These proceduresinclude temporomandibular joint dysfunction, wherein jaw movement islimited and may be painful.

Currently, these joint lesions are treated by opening the jointsurgically, or accessing the joint arthroscopically, and removing theadhesions. This treatment has the disadvantage of inducing furtherfibrosis during the healing process. The compositions and methods of thepresent disclosure inhibit subsequent fibrosis and adhesion formation inthe joint, thus increasing the chance of successful therapy.

Thoracic surgeries, including sternectomy which can be hazardous afterprimary surgery because of adhesion formation between the heart or aortaand sternum, bypass anastomosis, and heart valve replacement.

Cranial surgeries, including adhesions involving the skull, dura andcortex can complicate the secondary procedures.

Ocular surgeries, including strabismus surgery, glaucoma filteringsurgery, and lacrimal drainage system procedures.

Oral surgeries, including the treatment of temporomandibular jointdysfunction.

Other applications that may benefit from the inhibition of scarformation and fibrosis include the following implanted devices:nephrostomy tube, peritoneal drainage tube, artificial hip joint,artificial heart valve, peripheral nerve repair and other prostheses andintravenous catheter. Implants may be treated by coating or impregnatingthe implant, or a portion thereof, with a composition provided by thepresent disclosure. The present disclosure may also provide for animproved implant, in which the improvement comprises a coating on theimplant, which coating consists of a suitable amount of aninhibitory-adhesive composition.

The compositions and methods of the present disclosure can also befashioned into tissue adhesives and sealants which may be useful formedical and veterinary applications, including, but not limited to,wound closure, supplementing or replacing sutures or staples in internalsurgical procedures such as intestinal anastomosis and vascularanastomosis, tissue repair, preventing leakage of fluids such as blood,bile, gastrointestinal fluid and cerebrospinal fluid, ophthalmicprocedures, and drug delivery.

In one embodiment, the hydrogel tissue adhesive of the disclosure mayalso be used to bond at least two anatomical sites together. In thisembodiment, the first aqueous solution or dispersion is applied to atleast one anatomical site, and the second aqueous solution or dispersionis applied to at least one of either the same site or one other siteusing the methods described above. The two or more sites are contactedand held together manually or using some other means, such as a surgicalclamp, for a time sufficient for the mixture to cure. Alternatively, amixture of the two aqueous solutions or dispersions is applied to atleast one of the anatomical sites to be bonded using methods describedabove. The two or more sites are contacted and held together manually orusing some other means, such as a surgical clamp, for a time sufficientfor the mixture to cure.

In another embodiment, the aldehyde-functionalized dextran, and themulti-arm polyethylene glycol amine may be used in the form of finelydivided powders. The powders may be prepared using any suitable method.For example, each of the aqueous solutions or dispersions describedabove may be dried using heat, vacuum, a combination of heat and vacuum,or by lyophilization, to form powders. Optionally, the powders may becomminuted into finer particles using methods known in the artincluding, but not limited to, grinding, milling, or crushing with amortar and pestle. The finely divided powders may be sterilized usingthe methods described above. The finely divided powders may be appliedto an anatomical site on tissue of a living organism in a variety ofways. For example, the powders may be individually applied to the sitein any order by sprinkling or spraying. Additionally, the powders may bepremixed and the resulting mixture applied to the site by sprinkling orspraying. The powders may be hydrated on the site by the addition of anaqueous solution such as water or a suitable buffer (e.g.,phosphate-buffered saline) or by the physiological fluids present at thesite. The finely divided powders may also be used to bond two anatomicalsites together as described above for the aqueous solutions ordispersions. Alternatively, the powders may be hydrated with water or asuitable aqueous solution prior to use to form the first and secondaqueous solutions or dispersions, described above.

The disclosure of all references cited in the present disclosure areherein incorporated by reference in their entirety.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

Reagent Preparation

Preparation of Dextrans Having Pendant Aldehyde Groups (AFD-15-177-115%and AFD-10-216-94%)

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 10 kDa to about 15 kDa, an equivalent weightper aldehyde group of about 177, and a degree of aldehyde substitutionof about 115% was prepared using a two-step procedure. In the firststep, dextran having an average molecular weight of about 8.5-11.5 kDawas reacted with glycidol to form alkylated dextran. In the second step,the alkylated dextran was oxidized with sodium periodate to oxidize theterminal diol groups added in the first step to give dextran havingpendant aldehyde groups.

In the first step, 350 g of dextran (average molecular weight of about8.5-11.5 kDa, Sigma-Aldrich, Milwaukee, Wis.) was suspended in 385 mL ofwater and heated to 55° C. To this solution was added 437 mL of sodiumhydroxide solution (20 wt % in water), followed by the slow addition(4.5 mL/min) of glycidol (637 g, Aldrich) over a 2 hour period at 55° C.Then, the mixture was maintained at 55° C. for an additional 2 hours,after which the reaction mixture was cooled to room temperature andallowed to stir slowly for an additional 12 hours. The resulting yellowhomogeneous mixture was neutralized with 50% HCl (final pH was 6.6). Thesample was precipitated in approximately 5× volume of cold isopropanol(˜0° C.). The isopropanol layer was decanted off, the solid productwashed with cold isopropanol, and the process of dissolution followed byprecipitation was repeated two more times. Three hundred grams of thecrude material (containing roughly 75 g of isopropyl alcohol) wastransferred to a round bottom flask and rotovapped for approximately 2hours to remove excess isopropyl alcohol. The material was dissolved in2 L of deionized water and purified on a TFF system (tangential flowfiltration column), with a molecular weight cutoff of 3,000 MW(Millipore Corp., Billerica, Mass.). The sample was run on the TFFsystem with 7 exchanges; 219 g of material was recovered.

In the second step, 219 g of the solid product from the first step wasdissolved in 2,025 mL of water in a round bottom flask and then theresulting solution was cooled to 7-8° C. Sodium periodate solution(2,190 g in 2,354 mL of water) was added to the round bottom flaskdropwise over 60 min, the reaction mixture was stirred an additional 30min after the addition was completed, and then cooled to 0° C. toprecipitate residual sodium periodate and filtered. The filtrate wascollected in a 12 L multineck flask. To the filtrate was added 158.4 gof calcium chloride followed by addition of 107.3 g of potassium iodide,resulting in the formation of a reddish brown solution, which wasstirred for 20 min. To this mixture, acetone was added at 3× volume toprecipitate the solids and the mixture was stirred for an additional 30min. The solids were collected by vacuum filtration and washed withadditional acetone (approximately 1 L). The washed precipitate was driedunder vacuum, and about 254 g of an off-white solid material wasrecovered.

The product was dissolved in water to about a 12 wt % solution andpurified using a TFF system with a 1000 MW cutoff membrane. After 18volume exchanges, the product was lyophilized to dryness, yielding 100 gof a cream colored solid.

The equivalent weight per aldehyde group of the product was determinedby titration of the hydroxylamine adduct using the method described byZhao and Heindel (Pharmaceutical Research 8:400, 1991). Specifically,the equivalent weight (EW) per aldehyde group was determined as follows.The sample was dissolved in water to give a 20 wt % solution. To thissolution was added 25 mL of hydroxylamine hydrochloride solution. Theresulting mixture was vortexed briefly and then allowed to stand at roomtemperature for 2 hours. After that time, the solution was titrated withstandardized sodium hydroxide solution (0.25 N) until the color of thesolution changed from red to yellow, or to that of the startinghydroxylamine hydrochloride solution. Two replicate determinations weredone. The equivalent weight per aldehyde group was calculated using thefollowing formula: (Vol in mL of NaOH×N NaOH)×10⁻³ mol/weight ofsample)=1/EW. The equivalent weight per aldehyde group was determined tobe 177. The degree of aldehyde substitution was calculated assuming thata substitution of 100% corresponds to one pendant aldehyde group perdextran molecule and that the chemical structure of thealdehyde-functionalized dextran is:

The equivalent weight per aldehyde group of this structure is 204. Thedegree of aldehyde substitution of the aldehyde-functionalized dextranwas then calculated using the following formula: Degree of aldehydesubstitution=204/EW (determined as described above)×100. The degree ofaldehyde substitution was 115%. The resulting aldehyde-functionalizeddextran is referred to herein as AFD-15-177-115% (AFD-MW-EW-degree ofaldehyde substitution).

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 10 kDa to about 15 kDa and an equivalentweight of about 216 and a degree of aldehyde substitution of about 94%was prepared using the same general method as described above forAFD-15-177-115%. The resulting aldehyde-functionalized dextran isreferred to herein as AFD-10-216-94%.

Preparation of Oxidized Dextran (D10-50)

Dextran aldehyde was made by oxidizing dextran having a weight-averagemolecular weight of about 8.5-11.5 kDa (Sigma-Aldrich) in aqueoussolution with sodium metaperiodate. The oxidized dextran, referred toherein as D10-50, had an average molecular weight of about 10,000 Da andan oxidation conversion of about 50% (i.e., about half of the glucoserings in dextran are oxidized to dialdehydes). The oxidation conversionof the oxidized dextran was determined by proton NMR to be about 50%(equivalent weight per aldehyde group=146). In the NMR method, theintegrals for two ranges of peaks are determined, specifically, —O₂CHx-at about 6.2 parts per million (ppm) to about 4.15 ppm (minus the HODpeak) and —OCHx- at about 4.15 ppm to about 2.8 ppm (minus any methanolpeak if present). The calculation of oxidation level is based on thecalculated ratio (R) for these areas, specifically, R═(OCH)/(O₂CH).

Preparation of Eight-Arm PEG 10K Octaamine (P8-10-1)

Eight-arm PEG 10K octaamine (M_(n)-10 kDa) is synthesized using thetwo-step procedure described by Chenault in commonly owned U.S. Pat. No.7,868,132. In the first step, the 8-arm PEG 10K chloride is made byreaction of thionyl chloride with the 8-arm PEG 10K octaalcohol. In thesecond step, the 8-arm PEG 10K chloride is reacted with aqueous ammoniato yield the 8-arm PEG 10K octaamine. A typical procedure is describedhere.

The 8-arm PEG 10K octaalcohol (M_(n)=10000; NOF SunBright HGEO-10000),(100 g in a 500-mL round-bottom flask) is dried either by heating withstirring at 85° C. under vacuum (0.06 mm of mercury (8.0 Pa)) for 4hours or by azeotropic distillation with 50 g of toluene under reducedpressure (2 kPa) with a pot temperature of 60° C. The 8-arm PEG 10Koctaalcohol is allowed to cool to room temperature and thionyl chloride(35 mL, 0.48 mol) is added to the flask, which is equipped with a refluxcondenser, and the mixture is heated at 85° C. with stirring under ablanket of nitrogen for 24 hours. Excess thionyl chloride is removed byrotary evaporation (bath temp 40° C.). Two successive 50-mL portions oftoluene are added and evaporated under reduced pressure (2 kPa, bathtemperature 60° C.) to complete the removal of thionyl chloride. ProtonNMR results from one synthesis are: ¹H NMR (500 MHz, DMSO-d6) δ3.71-3.69 (m, 1611), 3.67-3.65 (m, 16H), 3.50 (s, ˜800H).

The 8-arm PEG 10K octachloride (100 g) is dissolved in 640 mL ofconcentrated aqueous ammonia (28 wt %) and heated in a pressure vesselat 60° C. for 48 hours. The solution is sparged for 1-2 hours with drynitrogen to drive off 50 to 70 g of ammonia. The solution is then passedthrough a column (500 mL bed volume) of strongly basic anion exchangeresin (Purolite® A-860, The Purolite Co., Bala-Cynwyd, Pa.) in thehydroxide form. The eluant is collected and three 250-mL portions ofde-ionized water are passed through the column and also collected. Theaqueous solutions are combined, concentrated under reduced pressure (2kPa, bath temperature 60° C.) to about 200 g, frozen in portions andlyophilized to give the 8-arm PEG 10K octaamine, referred to herein asP8-10-1, as a colorless waxy solid.

Preparation of 8-Arm PEG 10K Hexadecaamine (P8-10-2)

An 8-arm PEG 10K hexadecaamine, referred to herein as “P8-10-2”, havingtwo primary amine groups at the end of the arms, was prepared using atwo-step procedure, as described by Arthur in U.S. Pat. No. 8,282,959,in which 8-arm PEG 10K was reacted with methanesulfonyl chloride indichloromethane in the presence of triethylamine to produce 8-arm PEG10K mesylate, which was subsequently reacted withtris(2-aminoethyl)amine to give the 8-arm PEG 10K hexadecaamine. Atypical synthesis is described here.

To a solution of 10 g of 8-arm PEG 10K (M_(n)=10,000; NOF, Tokyo, Japan)in 50 mL of dichloromethane stirred under nitrogen and cooled to 0° C.is added 2.2 mL of triethylamine, followed by 1.2 mL of methanesulfonylchloride. The mixture is allowed to warm to room temperature and isstirred overnight. The reaction mixture is transferred to a separatoryfunnel and washed gently three times with 15 mL portions of 1 Mpotassium dihydrogen phosphate, followed by 15 mL of 1 M potassiumcarbonate, and then 15 mL of water. The dichloromethane layer is driedover magnesium sulfate, filtered, and concentrated by rotary evaporationto afford 11.17 g of 8-arm PEG 10K mesylate.

A mixture of 10 g of 8-arm PEG 10K mesylate and 45 mL oftris(2-aminoethyl)amine dissolved in 45 mL of water is stirred at roomtemperature for 24 hours. The reaction mixture is diluted with 45 mL of5% (w/w) aqueous sodium bicarbonate and extracted with a total of 500 mLof dichloromethane divided in 3 portions. The dichloromethane solutionis dried over sodium sulfate, and concentrated by rotary evaporation to20-25 g. Ether (100 mL) is added to the concentrated dichloromethanesolution with vigorous stirring, and the mixture is cooled to 0° C.,causing a waxy solid to separate from solution. The solvent is decantedfrom the waxy solid, and the waxy solid is dried under vacuum to givethe 8-arm PEG 10K hexadecaamine (P8-10-2).

Examples 1-5

Low Swell Hydrogels

The purpose of these Examples was to demonstrate the low swell propertyof the hydrogels disclosed here.

Hydrogels were formed by mixing two aqueous solutions, the first aqueoussolution containing the aldehyde-functionalized dextran(AFD-15-177-115%) and 300 ppm of FD&C Blue #1 dye, and the secondaqueous solution containing a polyethylene glycol amine (P8-10-1). Thetwo aqueous solutions were mixed using a dual barrel syringe with a 16stage mixing tip (MEDMIX SYSTEMS AG, Rotkreuz, Switzerland). Theconcentrations of the aqueous solutions used are given in Table 1. Theresulting mixture was injected directly into a piece of plastic tubinghaving an internal diameter of about 4 mm. After gelation, the hydrogelwas removed from the tubing by slitting the tubing open. The resultingcylindrical hydrogel was cut into pieces having a length ofapproximately 10 mm and each piece was placed inside a separate piece ofplastic tubing, which had an internal diameter of about 5 mm and alength of about 20 mm. The length of each hydrogel piece was measuredusing a ruler to the nearest 0.5 mm, and then each piece of tubing wasplaced into a separate 20 mL glass vial containing phosphate-bufferedsaline (PBS). The vials were capped and the lids were sealed with tape.Then, the vials were placed in an incubator at 37° C. on their sides tomaintain the hydrogels in a horizontal position. The vials were removedfrom the incubator at intervals, the length of the hydrogels wasmeasured as described above, and then the vials were returned to theincubator.

The results are summarized in Table 1 as the mean and standard deviationof the swell, where the swell is the percentage increase in the lengthof the hydrogel relative to its initial length. The negative swellvalues in the table indicate that the hydrogel decreased in length. Allcompositions of the present disclosure exhibit “low swell” as definedherein as having a % swell of less than about 2% as measured by thistechnique.

TABLE 1 Swell of Hydrogels Aldehyde- Functionalized Multi-Arm DextranPEG amine Swell Example Solution solution Day (%) 1 AFD-15-177- P8-10-11 −17 ± 6  155% 15 wt % 2 −24 ± 10 10 wt % 4 −21 ± 14 7 −26 ± 11 10 −26± 11 14 −19 ± 15 28 −37 ± 16 2 AFD-15-177- P8-10-1 1   6 ± 8 115% 15 wt% 2   1 ± 3 20 wt % 4 −1 ± 5 7   0 ± 4 10 −7 ± 5 14 −5 ± 7 28 −6 ± 6 3AFD-15-177- P8-10-1 1   5 ± 6 115% 17.5 wt % 2   3 ± 5 15 wt % 4   8 ± 57   2 ± 3 10 −1 ± 2 14   2 ± 3 28   1 ± 6 4 AFD-15-177- P8-10-1 1 −6 ± 6115% 20 wt % 2 −4 ± 5 10 wt % 4   2 ± 9 7 −2 ± 9 10 −2 ± 9 14  −7 ± 1228 −25 ± 23 5 AFD-15-177- P8-10-1 1   0 ± 7 115% 15 wt % 2 −6 ± 6 15 wt% 4   0 ± 7 7 −3 ± 9 10 −3 ± 9 14  −4 ± 12 28 −11 ± 7 

Comparative Examples 1-6

Hydrogels were prepared as described in Comparative Examples 1-6 using afirst aqueous solution comprising AFD-15-177-115% or an oxidized dextran(D10-50), prepared as described above, and a second aqueous solutioncomprising P-8-10-1 or a mixture of P8-10-1 and P-8-10-2, as indicatedin Table 2. In addition, hydrogels were formed using a commerciallyavailable product, DuraSeal™ Dural Sealant (COVIDEAN, Mansfield, Mass.).The swell of the hydrogels was measured as described in Examples 1-5.The results are presented in Table 2.

TABLE 2 Swell of Comparative Hydrogels Multi-Arm Comparative Dextran PEGamine Swell Example Solution solution Day (%) 1 AFD-15-177- P8-10-1 1 11± 1  115% 20 wt % 2 15 ± 7  20 wt % 4 15 ± 7  7 14 ± 5  2 AFD-15-177-P8-10-1 1 37 ± 10 115% (15 wt %)/ 2 40 ± 11 15 wt % P8-10-2 6 42 ± 10 (5wt %) 3 AFD-15-177- P8-10-1 1 39 ± 8  115% (15 wt %)/ 2 39 ± 8  20 wt %P8-10-2 6 33 ± 9  (5 wt %) 4 AFD-15-177- P8-10-1 1 84 ± 7  115% 30 wt %2 74 ± 12 15 wt % 6 85 ± 10 5 AFD-15-177- P8-10-1 1 76 ± 5  115% 40 wt %2 81 ± 7  15 wt % 6 87 ± 9  6 NA NA 1 48 ± 5  DuraSeal ™ 2 37 ± 7  6 47± 5 

As can be seen by comparing the data in Table 1 with the data in Table2, the hydrogels disclosed herein have significantly lower swell thanthe comparative hydrogels.

Example 6

In Vivo Adhesion Prevention in a Rabbit Laminectomy Model

The compositions of the present disclosure were tested to determinetheir effectiveness in preventing the formation of deleterious fibroustissues in a Rabbit Laminectomy study. Two low swell compositions (i.e.,Examples 1 and 5) were evaluated for adhesion prevention in a rabbitdorsal laminectomy model.

Aqueous solutions of AFD-15-177-115% (10 wt % and 15 wt %) and P8-10-1(15 wt %) were prepared and sterilized as follows. The appropriateweight of each component was calculated and adjusted for moisturecontent (as measured using a Mettler Toledo HB43-S moisture analyzingbalance). This amount was weighed into a glass vessel and the calculatedweight of water was added. A volume of a stock solution of FD&C Blue #1dye was added to each of the AFD-15-177-115% solutions to give a finalconcentration of 300 ppm. The resulting solutions were capped and placedon a shaker incubator at 170 rpm and 40° C. until the components wherefully dissolved. These solutions were then filtered through 0.45 μmmembrane filters and stored at room temperature until syringe fill.

Twelve dual barreled, 5 mL syringes were prepared by placing pistons inthe bottom of each syringe and using the appropriate plunger to evenlyposition the plungers in the barrels. The syringes (12), plungers (12),caps (12) and 16-step tapered mix tips (30) (MEDMIX SYSTEMS AG) wereplaced into sterilization pouches and steam sterilized in an autoclaveset to a standard “hard goods” cycle. Also sterilized were 4 blunt endedleur-type stainless steel transfer cannulas and two sets ofsterilization pouches and labels for packaging. A biological hood wasprepared for the sterile fill procedure by thoroughly wiping down allsurfaces with a disinfectant and exposing all of the cabinet innersurfaces to UV light for 30 min. The bottles containing the aqueoussolutions were sprayed and wiped down with 100% ethanol prior totransfer into the biological hood. These solutions were sterile filteredthrough pre-sterilized 0.2 μm filters into pre-sterilized containers.The pre-sterilized syringes and packaging materials were thentransferred into the biological hood. Six syringes were filled with eachsolution. The AFD-15-177-115% solution was filled first into theun-notched side of the dual barreled syringes and then the P8-10-1solution was filled into the notched side of the syringe. The syringeswere capped, labeled, and placed in a pre-sterilized pouch with aplunger and two or three of the 16-step mix tips and the pouch wassealed. Each primary pouch was then labeled and placed into a secondarypre-sterilized pouch and that pouch was sealed. This procedure provideda system where the primary sterile pouch can be placed onto the sterilesurgical field from the secondary pouch without compromising the sterilefield. All materials were stored at room temperature until use.

Twelve New Zealand White Rabbits were used in the study, six for each ofthe two compositions tested. Each rabbit had two laminectomy sites, L2 &L4, one treated with one of the low swell hydrogel compositions and oneuntreated control. The treatment site location was randomized withineach treatment group. The laminectomy created an approximately 5 mm×10mm exposure of the spinal cord. A mixture of the first aqueous solutioncontaining AFD-15-177-115% and the second aqueous solution containingP8-10-1 was applied to cover the defect at the treatment site using adual barrel syringe with a 16 stage mixing tip. The average applicationdose was 0.08 g. At necropsy after 28 days, each site was examined andgraded for adhesions with the following scales:

Adhesion Extent of Total Area. 0: None (no adhesions); 1: 1-25%; 2:26-50%; 3: 51-75%; 4: 76-100%. The adhesion extent is the amount of theoriginal Laminectomy area covered with scar tissue.

Adhesion Severity Scoring. 0: None (no adhesions); 1: One thin filmyadhesion, non-adherent; 2: Definite adhesions, blunt dissectionrequired; 3: Dense adhesions, sharp dissection required.

Two control sites and three treated sites had boney tissue growth overthe defect site, and were therefore unable to be graded for adhesions.All tissues were collected and sent for pathological and histologicalevaluation.

ANOVA analysis of the Adhesion Total Score (AD_TS=AdhesionSeverity+Adhesion Extent), versus group and site showed that both lowswell hydrogel compositions gave equal results and both treatment siteswere equivalent. Therefore, the scores obtained with both compositionswere combined. All the control sites had dense adhesions to the spinalcord with AD_TS≧6.

The ANOVA analysis results for combined treated sites and control sitesversus the responses of adhesion extent, adhesion severity and adhesiontotal score are shown in Table 3. The combined treated sites were 56%adhesion free, 22% had AD_TS≦2 and the last 22% had AD_TS≧6. The averageadhesion severity and extent scores had reductions over the controlgroup of 70 and 71%, respectively. These results are surprisinglysuperior to the published results with existing commercial DuraSeal™(e.g., Mo et al., “Evaluation of Perivascular Adhesion Formation in NewZealand White Rabbits Using Oxiplex and DuraSeal Xact Adhesion BarrierSystem” SAS Journal, 3(2), 76, June 2009).

TABLE 3 ANOVA Analysis of Adhesions Treated Sites Control Sites Ave ±Std Dev Ave ± Std Dev Response (n = 10) (n = 11) P Value Adhesion Extent0.9 ± 1.3 3.0 ± 0   0.000 (0-4) Adhesion Severity 1.0 ± 1.5 3.4 ± 0.960.001 (0-3) Adhesion Total 1.9 ± 2.8 6.4 ± 1.0  0.000 Score

The hemostatic properties of the low swell hydrogel compositionsdisclosed herein were also demonstrated in this study with rabbit no. 8.This rabbit bled severely during the first (L2) laminectomy. The surgeonwas going to sacrifice this rabbit but instead, a mixture of the firstand second aqueous solutions were applied to the laminectomy site attwice the dose used in the other rabbits. The bleeding stopped to theextent that the animal survived and the second laminectomy site wascompleted as a control site. This treatment site showed no signs ofadhesions at 28 days.

Example 7

Cytotoxicity Testing

The two compositions described in Examples 1 and 5 were tested forcytotoxicity using dilutions of the component aqueous solutions andextracts from the hydrogel using three cell lines.

The cell lines (purchased from American Type Culture CollectionManassas, Va.) used in the cytotoxicity testing were L929 (ATCC#CCL-1)cell line as per ISO 109933; MG63 (ATCC#CRL-1427) osteosarcoma derivedcell line; and S16 (ATCC#CRL-2941) nerve cell line derived from Schwanncells.

Cell Preparation/Cell Maintenance

Cells were cultured in T-75 (75 cm²) flasks containing 10 mL of theappropriate medium (Eagle's Minimum Essential Medium plus 10% horseserum and penicillin/streptomycin for L929; Eagle's Minimum EssentialMedium plus 10% heat-inactivated fetal bovine serum andpenicillin/streptomycin for MG-63; and Dulbecco's Modified Eagle'sMedium plus 10% fetal bovine serum for S16). After reaching 80-90%confluence, the cells were sub-cultured into new T-75 flasks at thesuggested splitting ratio.

Preparation of Assay Plates (96 Well) Containing Cells

For each flask, the medium was removed and the cell layer was washedwith 5 mL of PBS (phosphate-buffered saline). The PBS was discarded and1.5 mL of trypsin was added per flask. The trypsin was gently rocked inthe flask to allow equal dispersion across the cell layer until thecells were detached from the flask surface. To the trypsin-cellsuspension, 8.5 mL of complete medium was added to inactivate thetrypsin and provide a homogeneous cell suspension. The suspension wasplaced into a sterile 15 mL tube and centrifuged at 130×g for 10 min.After centrifugation, the medium was removed and 10 mL of fresh mediumwas added to the tube. The cells were re-suspended by mild titrationuntil a homogeneous suspension was obtained.

To a clean 1.5 mL microfuge tube, 300 μL of PBS, 500 μL 0.4% Trypan BlueStain solution, and 200 μL of the cell suspension were added and theresulting suspension was mixed thoroughly and then allowed to stand for5 min.

A hemocytometer (Hausser Scientific, Horsham, Pa.) was set up with thecover glass in place. The cell suspension was mixed and an aliquot wasadded to the hemocytometer, which was then placed onto the stage of amicroscope and the number of cells was counted in each of the eightblocks. The number of cells per mL of suspension was calculated byaveraging the number of cells in the eight blocks and multiplying by5×10⁶. To obtain the volume of the cell suspension to add to each well,the number of cells desired for each well was divided by the number ofcells per mL of cell suspension. Fresh medium was added to each well ofthe 96-well plate (volume of fresh medium=200 μL−volume of cellsuspension). Next, the volume of cell suspension to give the proper cellnumber per well for each cell type was added to each well (i.e., forL929, 25,000 cells/well; for MG63, 2,000 cells/well; and for S16, 15,000cells/well) and the plates were incubated for 24 h at 37° C. with 5%CO₂.

Sample Preparation for Soluble Components

Samples of AFD-15-177-115% and P8-10-1, 100 mg each, were weighed intoseparate clean glass vials (20 mL) and the final weight was brought to1.0 g with sterile water, giving a final of concentration of 10 wt %.The vials were capped and placed in a 37° C. shaker at 170 rpm for 1hour to dissolve the samples. When the samples were completelydissolved, the solutions were sterile filtered into sterile glass vialsusing a 0.2 μm syringe filter. Dilutions of these solutions wereprepared using the complete medium of each cell type to be assayed witha minimum final volume of 800 μL or enough to allow for 200 μL/well witha minimum of 200 μL excess. The dilutions were made in sterile microfugetubes to yield concentrations of 10, 5, 2.5, 1, 0.5, 0.1, 0.05 and 0mg/mL.

Hydrogel Samples for MEM (Minimum Essential Medium) Elution

Hydrogels were produced using sterile double barrel syringes fitted witha six stage mixer and 1 mL of each sterile aqueous solution in thesyringe (i.e., AFD-15-177-115% (10 wt %) and P8-10-1 (15 wt %)) inseparate barrels of one syringe and AFD-15-177-115% (15 wt %) andP8-10-1 (15 wt %) in separate barrels of a second syringe. The hydrogelwas formed by dispensing the mixed aqueous solutions (1.0 g) from thedouble barrel syringe into a sterile glass slide mold. The hydrogelformed was extracted with MEM (a ratio of 0.1 g gel/mL MEM) at 37° C.and 15 rpm for 24 h. The extracted MEM was placed in sterile tubes anddiluted with MEM to give extract percentages of 90, 75, 50, 25, 12.5,6.25, and 0. A volume of 200 μL was added to each PBS washed wellcontaining cells in triplicate following the assay protocol describedbelow.

Assay Procedure

The 96-well plates were removed from the incubator and placed in abiological hood. The medium was removed from the wells and the celllayer was washed 3 times with PBS. After the washes, the PBS was removedand 200 μl of the designated sample was added to the well in triplicate.The plates were then placed back into the incubator and cultured for 48hours.

Cell Proliferation Assay Using Tetrazolium Dye

The 96-well plates containing cells were placed in a biological hood andthe medium was removed. The cells were then carefully washed 3 timeswith sterile PBS. After the last wash when the PBS was removed, 200 μLof sample dilutions were added to the wells in triplicate. The plateswere covered and returned to the incubator for 24 hours. At the end ofthe incubation period, the plates were removed from the incubator andpictures were taken of representative wells containing the samples andthe controls. Then, the medium was removed and the cells were washed 3times with PBS. Phenol-Red free medium (100 μL) and then 10 μL of WST8reagent (Cayman Chemical Company, Ann Arbor, Mich.) was added to eachwell. The solution in the wells was gently mixed by tapping the plateand the plate was returned to the incubator for 2 hours to allow thereaction to occur. At the end of the reaction period, the plate wasremoved from the incubator and 90 μL of the well contents wastransferred to a clean 96-well plate. The plate was placed into aspectrophotometer and the absorbance was read at 450 nm.

The results are shown in Tables 4-6. In the ISO testing with L929 cells,all the gel formulations passed with undiluted extracts (see Table 4).The AFD-15-177-115% component was only toxic at very high concentrationswhich are not obtainable in the formed hydrogels. The MG63 cell line washighly tolerant to all the hydrogel components, as shown in Table 5. TheS16 cell line was the most sensitive cell line. The results with the S16cell line (Table 6) indicated that lowering the AFD-15-177-115% contentin the composition lowers the risk of negatively affecting nerve cellsexposed to these compositions.

TABLE 4 Cell Viability of L929 Cells with Soluble Components andHydrogel Extracts Hydrogel Extract P8-10-1 P8-10-1 (15 wt %)/ (15 wt %/Soluble Component AFD-15-177- AFD-15177- AFD-15-177- 115% 115% mg/mLP8-10-1 115% (10 wt %) (15 wt %) 10 105 ± 7   12 ± 1  98 ± 2 98 ± 8 5106 ± 4   88 ± 2 104 ± 5 104 ± 9  2.5 111 ± 7  102 ± 1 107 ± 8 110 ± 9 1 104 ± 6   96 ± 3 100 ± 2 102 ± 7  0.5 100 ± 13 102 ± 4 108 ± 5 110 ±8  0.1 111 ± 8  112 ± 4 118 ± 5 119 ± 13 0.05 107 ± 2  102 ± 3 111 ± 8105 ± 12 0 100 ± 0  100 ± 0 100 ± 0 100 ± 0 

TABLE 5 Cell Viability of MG16 Cells with Soluble Components andHydrogel Extracts Hydrogel Extract P8-10-1 P8-10-1 (15 wt %/ (15 wt %/Soluble Component AFD-15-177- AFD-15-177- AFD-15-177- 115% 115% mg/mLP8-10-1 115%8 (10 wt %) (15 wt %) 10 100 ± 7   97 ± 11  79 ± 8  74 ± 115 107 ± 13 107 ± 6   89 ± 5  81 ± 17 2.5 113 ± 15 114 ± 4   97 ± 4  89 ±11 1 116 ± 16 114 ± 7  102 ± 7  97 ± 20 0.5 118 ± 9  124 ± 3  106 ± 3 98 ± 21 0.1 110 ± 8  114 ± 1  102 ± 4  94 ± 22 0.05 99 ± 6 101 ± 1   93± 3  84 ± 13 0 100 ± 0  100 ± 0  100 ± 0 100 ± 0 

TABLE 6 Cell Viability of S16 Cells with Soluble Components and HydrogelExtracts Hydrogel Extract P8-10-1 P8-10-1 (15 wt%)/ (15 wt %/ SolubleComponent AFD-15-177- AFD-15-177- AFD-15-177- 115% 115% mg/mL P8-10-1115% (10 wt %) (15 wt %) 10  97 ± 12  16 ± 1  92 ± 4 60 ± 9 5 103 ± 9  16 ± 0  96 ± 4 32 ± 8 2.5 102 ± 9   59 ± 0 102 ± 5  32 ± 20 1 100 ± 2  80 ± 2 114 ± 4 103 ± 1  0.5 100 ± 4   94 ± 2 120 ± 5 112 ± 2  0.1 98 ±2  98 ± 1 119 ± 5 113 ± 2  0.05 96 ± 2  96 ± 4 113 ± 1 112 ± 2  0 100 ±0  100 ± 0 100 ± 0 100 ± 0 

Example 8

Delivery of Low Swell Composition for Use as a Dural Sealant-DorsalLaminectomy of Lumbar Vertebrae Model

The purpose of this study was to evaluate the delivery of a low swellcomposition of the disclosure to dorsal laminectomy sites in a cadavericovine model.

The low swell composition used in this Example was prepared using afirst aqueous solution containing AFD-10-216-94% at 10 wt % and 300 ppmof FD&C Blue #1 dye and a second aqueous solution containing P8-10-1 at15 wt %. The two aqueous solutions were mixed and delivered using a dualbarreled syringe equipped with an 8-stage mixing tip (both from MEDMIXSYSTEMS AG). The aqueous solutions were prepared and sterilized asdescribed in Example 6. Two 3-year old female sheep (Ovis aries) havingan average weight of about 70 g (obtained from Archer Farms, Inc.,Darlington, Md.) were used in the study.

Surgical Procedure

In this study, the lumbar vertebrae of the two sheep were isolated and adorsal laminectomy was performed on each. The laminectomy sites were 3cm and 4.5 cm. Lumbar vertebrae were isolated by dissection of thesurrounding musculature. Laminectomies were performed with Kerrisonrongeurs. The two aqueous solutions described above were first mixed anddelivered using the dual barreled syringe and mixing tip described abovelateral to the spinal cord by deviating the cord with a probe.Subsequently, the two aqueous solutions were mixed and delivered dorsalto the spinal cord filling the entire defect in the dorsal lamina.Following delivery of the solutions to dorsal laminectomy sites in sheepcadaveric lumbar vertebrae, the resulting hydrogel was allowed to setfor several minutes and then the vertebrae were cut in cross section attwo sites. The distribution of the hydrogel was recorded with digitalphotography and stored.

Results

The low swell composition and delivery system provided adequate workingtime for controlled delivery. Insertion of the syringe tip under thedorsal lamina allowed extension of the composition cranially andcaudally from the surgical site. The delivery of the composition wasenhanced by deviation of the spinal cord laterally with a blunt probe.This allowed the composition to distribute ventrally encompassing theentire circumference of the spinal cord. The results of this in vitrocadaver study showed that the low swell composition was delivered withthe syringe and mixing tip around the spinal cord.

Example 9

Non-Survival Sheep Laminectomy Model with Dural Nick

The purpose of this study was to validate the surgical procedure and thedelivery of a low swell composition of the disclosure to lumbarlaminectomy sites. The ability of the low swell composition of thedisclosure to seal small durotomies was also demonstrated.

The low swell composition used in this Example was the same as describedin Example 8. A skeletally mature female sheep (Ovis aries; age 5 years;weight 61.35 kg; from Archer Farms, Inc.) was used in this study.

Surgical Procedure

An intrathecal catheter was placed to measure CSF pressure and as anaccess portal to pressurize the subdural space. A dorsal laminectomy wasperformed on T13-L1, L2-L3 and L5-L6 using Kerrison rongeurs. A durotomywas performed at each site sequentially by making a small nick in thedura using an 18 gauge needle. A small amount of the low swellcomposition was applied to the dural nick at each laminectomy sitesequentially using the dual barreled syringe and allowed to set. Salinewith toluidine blue dye was injected into the catheter to assess leakageat the repair site. The procedures were video recorded. Following theleak check, the low swell composition was applied to the entire defectat each site taking care to fill the spinal canal around the cord viaslight retraction. The animal was euthanized at the conclusion of theprocedure under general anesthesia. Postoperative CT was performed ofthe lumbar spine. The lumbar spine was examined macroscopically atnecropsy and findings were recorded with digital photography.

Results

The results of this non-survival study showed that the low swellcomposition can be successfully delivered to dorsal laminectomy sites invivo. Small 18 gauge durotomies were successfully sealed, although therewere some inconsistencies due to the shape of the spinal cord and theviscosity of the low swell composition. The low swell composition wasalso successfully administered to the entire circumference of the spinalcord.

Example 10

Non-Survival Sheep Cranial Durotomy Model

The purpose of this study was to validate the surgical procedure and thedelivery of two low swell compositions of the disclosure. The ability ofthe low swell compositions to seal small durotomies was alsodemonstrated.

Two low swell compositions were evaluated in this study. The first lowswell composition was prepared using a first aqueous solution containingAFD-10-216-94% at 10 wt % and 300 ppm of FD&C Blue #1 dye and a secondaqueous solution containing P8-10-1 at 15 wt % (referred to herein asFormulation I). The second low swell composition was prepared using afirst aqueous solution containing AFD-10-216-94% at 15 wt % and 300 ppmof FD&C Blue #1 dye and a second aqueous solution containing P8-10-1 at15 wt % (referred to herein as Formulation II). The aqueous solutionswere prepared and sterilized as described in Example 6. The two aqueoussolutions were mixed and delivered using a dual barreled syringeequipped with an 8-stage, 12-stage, or 16-stage mixing tip (from MEDMIXSYSTEMS AG), as indicated in Table 7. A 6 year old female sheep (Ovisaries; weight 50.9 kg; from Archer Farms, Inc.) was used in this study.

Surgical Procedure

An intrathecal catheter was placed to measure CSF pressure and as anaccess portal to pressurize the subdural space. Four craniotomies (14 mmdiameter) using an Acra Cut DGR-II disposable cranial perforator(ACRA-CUT Inc. Acton, Mass.) were performed in the parietal bone. A 3-4mm long durotomy was made using an #11 blade at each site. A low swellcomposition was applied to each craniotomy site, as indicated in Table7. At the first two sites, a small amount of a low swell composition wasapplied to the durotomy and allowed to set. CSF pressure was increasedby Trendelenberg position. Leakage of CSF at the repair site wasassessed and recorded with video. A low swell composition was thenapplied to the entire craniotomy defect. At the second two sites, thelow swell composition was applied to the entire defect initially andleaks were assessed. The two formulations of low swell composition wereassessed at different sites and with each mixing tip (i.e., 8, 12 and16-stage) used. The animal was euthanized under general anesthesia atthe conclusion of the procedure. Postoperative cranial CT was performed.

Results

The results are summarized in Table 7.

TABLE 7 Administration of Low Swell Compositions at Each Craniotomy SiteCraniotomy Low Swell Site Formulation Amount Mixing Tip Observation Site1, I Small¹  8-stage Dural bleeding, Left Caudal no seal I Small 12-stage Dural bleeding, no seal I Half² 12-stage Dural bleeding, noseal II Small, 12-stage Dural bleeding, then Full³ hemorrhage tract Site2, II Small, 12-stage Dural seal, Left Cranial then Full  bleeding frombone hindered gelation of formulation Site 3, II Full 12-stage Duralseal, Right Rostral small amout iof bleeding from durotomy whichstabilized Site 4, II Full 16-stage Dural seal, Right Caudal largeramount of bleeding from durotomy which stabilized ¹2-4 drops of lowswell composition to cover durotomy ²Craniotomy filled half way with lowswell composition ³Craniotomy filled with low swell composition

The results of this non-survival study demonstrated that the low swellcompositions tested can be successfully delivered to cranial durotomysites in vivo. The craniotomy procedure allowed sufficient surface areato assess dural sealing competency. Small durotomies (2-3 mm) weresuccessfully sealed and challenged with increasing CSF pressure.Increased viscosity of applied low swell compositions applied with thegreater stage mixing tips allowed for more rapid adherence andcompensation for gravity. Gel time of the low swell compositions wasaffected by persistent bleeding from the dura or adjacent bone. Gelationof the low swell compositions in a dry environment appears to becritical to establish effective dural sealant properties. Gelation isinhibited by a moist environment from blood and CSF. Minimizing bloodand CSF at the application site allowed successful sealing of thedurotomy. In cases where a moderate amount of persistent hemorrhage fromeither the dura or the bone was observed, the low swell compositionswere still able to gel, creating an effective dural sealant whenchallenged with an increase in CSF pressure. The faster crosslinkingformulation (Formulation II) provided better results than the slowercrosslinking formulation (Formulation I). The number of stages in themixing tip also influenced the results with the higher staged mix tipsgiving better results than the lower stage mix tips. This correlates tothe increased mixing providing a gel with a higher crosslink density atthe time of application. Injection of dye into the intrathecal cathetercould not reach the cranial durotomy sites without meeting increasedresistance of injection pressure. The postoperative CT allowedvisualization of the craniotomy sites and the low swell compositions canbe distinguished between bone, air and fluid, but not soft tissue.

What is claimed is:
 1. A kit for forming a low swell hydrogel comprising: (a) a first aqueous solution or dispersion comprising one or more aldehyde-functionalized dextrans containing pendant aldehyde groups, said aldehyde-functionalized dextrans having a weight-average molecular weight of about 10,000 to about 20,000 Daltons and an equivalent weight per aldehyde group of about 226 to about 170; and (b) a second aqueous solution or dispersion comprising one or more polyethylene glycols having eight arms, substantially each arm of which is terminated with at least one primary amine group, wherein the polyethylene glycols have a number-average molecular weight of about 9,000 to about 11,000 Daltons; wherein (i) the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the first aqueous solution or dispersion is about 5 wt % to about 20 wt % and the total concentration of the polyethylene glycols in the second aqueous solution or dispersion is about 10 wt % to about 18 wt %; or (ii) the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the first aqueous solution or dispersion is about 5 wt % to about 10 wt % and the total concentration of the polyethylene glycols in the second aqueous solution or dispersion is about 10 wt % to about 20 wt %.
 2. The kit of claim 1, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 226 to about
 185. 3. The kit of claim 1, wherein the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the first aqueous solution or dispersion is about 5 wt % to about 10 wt % and the total concentration of the polyethylene glycols in the second aqueous solution or dispersion is about 10 wt % to about 18 wt %.
 4. The kit of claim 1, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 13,000 to about 17,000 Daltons.
 5. The kit of claim 1, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 222 to about
 189. 6. The kit of claim 1, wherein the polyethylene glycols have a number-average molecular weight of about 9,500 to about 10,500 Daltons.
 7. The kit of claim 1, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 15,000 Daltons and an equivalent weight per aldehyde group of about 177, wherein the polyethylene glycols have a number-average molecular weight of about 10,000 Daltons, and wherein the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the first aqueous solution or dispersion is about 10 wt % and the total concentration of the polyethylene glycols in the second aqueous solution or dispersion is about 15 wt %.
 8. The kit of claim 1, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 15,000 Daltons and an equivalent weight per aldehyde group of about 177, wherein the polyethylene glycols has a number-average molecular weight of about 10,000 Daltons, and wherein the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the first aqueous solution or dispersion is about 15 wt % and the total concentration of the polyethylene glycols in the second aqueous solution or dispersion is about 15 wt %.
 9. A dried hydrogel formed by a process comprising the steps of: combining in a solvent (a) one or more aldehyde-functionalized dextrans containing pendant aldehyde groups, said aldehyde-functionalized dextrans having a weight-average molecular weight of about 10,000 to about 20,000 Daltons and an equivalent weight per aldehyde group of about 226 to about 170, and (b) one or more polyethylene glycols having eight arms, substantially each arm of the which is terminated with at least one primary amine group, said polyethylene glycols having a number-average molecular weight of about 9,000 to about 11,000 Daltons, to form a low swell hydrogel; wherein (i) the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the solvent is about 5 wt % to about 20 wt % and the total concentration of the polyethylene glycols in the solvent is about 10 wt % to about 18 wt %; or (ii) the total concentration of the aldehyde-functionalized dextrans containing pendant aldehyde groups in the solvent is about 5 wt % to about 10 wt % and the total concentration of the polyethylene glycols in the solvent is about 10 wt % to about 20 wt %; and treating said hydrogel to remove at least a portion of said solvent to form the dried hydrogel.
 10. The dried hydrogel of claim 9, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 226 to about
 185. 11. The dried hydrogel of claim 9 wherein said dried hydrogel is in the form of a film.
 12. A composition comprising the reaction product of: a) at least one aldehyde-functionalized dextran containing pendant aldehyde groups, wherein the aldehyde-functionalized dextran has a weight-average molecular weight of about 10,000 to about 20,000 Daltons and an equivalent weight per aldehyde group of about 226 to about 170, and b) at least one polyethylene glycol having eight arms, substantially each arm of which is terminated with at least one primary amine group, wherein the polyethylene glycol has a number-average molecular weight of about 9,000 to about 11,000 Daltons; wherein (i) the composition contains about 5 wt % to about 20 wt % of the aldehyde-functionalized dextran and about 10 wt % to about 18 wt % of the polyethylene glycol; or (ii) the composition contains about 5 wt % to about 10 wt % of the aldehyde-functionalized dextran and about 10 wt % to about 20 wt % of the polyethylene glycol.
 13. The composition of claim 12, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 226 to about
 185. 14. A crosslinked hydrogel composition comprising: a) at least one aldehyde-functionalized dextran containing pendant aldehyde groups, wherein the aldehyde-functionalized dextran has a weight-average molecular weight of about 10,000 to about 20,000 Daltons and an equivalent weight per aldehyde group of about 226 to about 170, and b) at least one polyethylene glycol having eight arms, substantially each arm of which is terminated with at least one primary amine group, wherein the polyethylene glycol has a number-average molecular weight of about 9,000 to about 11,000 Daltons; wherein (i) the composition contains about 5 wt % to about 20 wt % of the aldehyde-functionalized dextran and about 10 wt % to about 18 wt % of the polyethylene glycol; or (ii) the composition contains about 5 wt % to about 10 wt % of the aldehyde-functionalized dextran and about 10 wt % to about 20 wt % of the polyethylene glycol; and wherein said aldehyde-functionalized dextran and said polyethylene glycol are crosslinked through covalent bonds formed between the pendant aldehyde groups of the dextran and the primary amine groups of the polyethylene glycol.
 15. The crosslinked hydrogel composition of claim 14, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 226 to about
 185. 16. A method for applying a low swell coating to an anatomical site on tissue of a living organism comprising the steps of: applying to the site (a) aldehyde-functionalized dextrans containing pendant aldehyde groups, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 10,000 to about 20,000 Daltons and an equivalent weight per aldehyde group of about 226 to about 170; followed by (b) polyethylene glycols having eight arms, substantially each arm of which is terminated with at least one primary amine group, wherein the polyethylene glycols have a number-average molecular weight of about 9,000 to about 11,000 Daltons, or (b) followed by (a), or premixing (a) and (b) and applying the resulting mixture to the site before the resulting mixture completely cures; and wherein the weight percent ratio of aldehyde-functionalized dextrans to polyethylene glycols is about 2:1 to about 1:4.
 17. The method of claim 16, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 226 to about
 185. 18. The method of claim 16, wherein the weight percent ratio of aldehyde-functionalized dextrans to polyethylene glycols is about 1:1 to about 5:18.
 19. The method of claim 16, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 13,000 to about 17,000 Daltons.
 20. The method of claim 16, wherein the aldehyde-functionalized dextrans have an equivalent weight per aldehyde group of about 222 to about
 189. 21. The method of claim 16, wherein the polyethylene glycols have a number-average molecular weight of about 9,500 to about 10,500 Daltons.
 22. The method of claim 16, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 15,000 Daltons and a degree of aldehyde substitution of about 108%, wherein the polyethylene glycols have a number-average molecular weight of about 10,000 Daltons, and wherein the weight percent ratio of aldehyde-functionalized dextrans to polyethylene glycols is about 1:1.5.
 23. The method of claim 16, wherein the aldehyde-functionalized dextrans have a weight-average molecular weight of about 15,000 Daltons and a degree of aldehyde substitution of about 108%, wherein the polyethylene glycols has a number-average molecular weight of about 10,000 Daltons, and wherein the weight percent ratio of aldehyde-functionalized dextrans to polyethylene glycols is about 1:1.
 24. The method of claim 16, wherein the site is tissue involved in or affected by a surgical procedure.
 25. The method of claim 24, wherein the surgical procedure is lumbar laminectomy, laminotomy, discectomy, flexor tendon surgery, spinal fusion, joint replacement or repair, abdominal procedures, gynecological procedures, musculoskeletal surgeries, thoracic surgeries, cranial surgeries, ocular surgeries, oral surgeries or implants. 